CA2132094A1 - Oligonucleotide modulation of protein kinase c - Google Patents
Oligonucleotide modulation of protein kinase cInfo
- Publication number
- CA2132094A1 CA2132094A1 CA002132094A CA2132094A CA2132094A1 CA 2132094 A1 CA2132094 A1 CA 2132094A1 CA 002132094 A CA002132094 A CA 002132094A CA 2132094 A CA2132094 A CA 2132094A CA 2132094 A1 CA2132094 A1 CA 2132094A1
- Authority
- CA
- Canada
- Prior art keywords
- pkc
- seq
- oligonucleotide
- dna
- oligonucleotides
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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Abstract
Compositions and methods are provided for the treatment and diagnosis of diseases associated with protein kinase C.
Oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding PKC. Oligonucleotides specifically hybridizable with a translation initiation site, 5'-untranslated region or 3'-untranslated region are provided. Oligonucleotides specifically hybridizable with a particular PKC isozyme or set of isozymes are also provided. Methods of treating animals suffering from disease amenable to therapeutic intervention by modulating protein kinase C expression with an oligonucleotide specifically hybridizable with RNA or DNA corresponding to PKC are disclosed.
Oligonucleotides are provided which are specifically hybridizable with nucleic acids encoding PKC. Oligonucleotides specifically hybridizable with a translation initiation site, 5'-untranslated region or 3'-untranslated region are provided. Oligonucleotides specifically hybridizable with a particular PKC isozyme or set of isozymes are also provided. Methods of treating animals suffering from disease amenable to therapeutic intervention by modulating protein kinase C expression with an oligonucleotide specifically hybridizable with RNA or DNA corresponding to PKC are disclosed.
Description
2~3 2~32~ PC~/V~93~0~213 OLIGON~C~OTID~ ~ODahATION O~ PROTEIN ~INA8~ C `-FIE~D O~ TE~ INVEN~IO~ ~ :
Thi~ invention relates to therapies, diagnostics, and ;:
research reagents for disease states which respond to 5 modulation of the expression of protein kinase C. In .:
particular, this invention relates to antisense oligonucleotides specifically hybridizable with nucleic acids relating to protein kinase C. These oligonucleotides have been ~ound to modulate the expression of protein kinase C.
10 Palliation and therapeutic effect result.
.
,, BX~XGRO~ND OF T~ I ~ B~TIO~
The phosphoryla~:ion of proteins plays~;a key role. in the transduction of: extracellular ~ignals into the cell. ~he enzymes, called kinases, which effect such phosphorylations are 15 targetæ for the ac~ion of growth factors, hormones, and other agents involved in cellular~ metabolism, proliferation and di~ferentiation. O~e o~ the major signal transduction pathways , .
::~ involves ~h~ enzyme protein kinase C (PKC), which is known to have a critical~ influence on cell prolifera~ion and 20 diflferen~iatIon. /PKC is activated by:diacylglyc~rolsl(DAG~s), . which are:m~tabolites released in signaI transduction.
Interest in PKC:was s~imulated by the finding that PKC
is the maj~r, and perhaps only, cellular ~eceptor through which `: a ~lass o~ tumor-p~omoting agents called phoxbol esters exert Z:5~: their pleiotropic effec~s; on~cells. Gescher et~al., Anti~
: Cancer~ ~Drug Desi~n 1989~, 4, 93~-1050 Phorb~ls~capable of tum~r production~ can mimic the effect of DAG in activa~ing PKC, :, ~ : , :
W093/19203 P~T~US93/02213 2 1 3 2 09 l _ 2 - ! r suggesting that these tumor promotors act through PKC and that activation of this enzyme is at least partially responsible for the resul~ing tumorigenesis. Parker et al., Science 1986, 233, 853-866.
Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestlnal tract convert lipids to DAG, thus activating PKC and altering cell:proliferation. This may explain the correlation between high dietary fat and colon cancer;. Weinstein, Cancer Res4 ~Suppl.) 1991, 51, 5080s~5085s.
It has also been demonstrated that a greater proportion of the PKC in th~ colonic mucosa of patients with colorectal cancer is _ in an activated state compared to that of patients without cancer. Sakanoue et al., Int . J. Can er 1991, 48, 803-806.
Increased tumorigenicity is also correlated with overexprèssion of PKC in cultured cells inoculated into nude . .
mice.~ A mutant ~orm of~ PXC induces highly malignant tumor cells with increased metastatic potential. Sphingoslne and related~ inhibitors of PKC activity have been ~hown to inhibit 20; tumor cell growth and radiation-induced transformation in vivo.
Endo et al., Cancer~Research 1991, 51, 1613-1618; Borek et al., P~oc. Natl. Acad. Scl. 1991, 88, 1953-1957. A number of experimental or clinically useful anti-cancer drugs show ~modulatory effects~on~PKC. Therefore,~inhibitors of PKC may be 25~ important cancer-preventive or therapeutic agents. PKC has been suggested as~a~plausible tar~et for~more rational design of con~entional anti-cancer drugs. Gescher, A. an~ Dale, I.L., :
Anti-Cancer Drug Design~ 198'~, ~, 93-105.
xperiments also indicate tha~ PKC plays an important 3G ~role~ in the pathophysiology of hyperproliferati~e skin ` disorders such~as~psoriasis~and skin cancer. ~Psoriasis is characterized~by~inflamma~ion,~ hyperproliferation ~of ~the epider~is~ nd~decreased~ dif~feréntiation~of~cells.~ Various studies indicate~a~role for PKC in causing~these symptoms. PKC
35~ stimulation in~çultured~keratinocytes can be shown ~o cause hyperproli~feration.~ ~Inflammation can be induced by phorbol esters ~and~ is regulated~by~P~C. DAG is implicated in ~he W093/19203 213 2 0 9 ~ PCT/US93/02213
Thi~ invention relates to therapies, diagnostics, and ;:
research reagents for disease states which respond to 5 modulation of the expression of protein kinase C. In .:
particular, this invention relates to antisense oligonucleotides specifically hybridizable with nucleic acids relating to protein kinase C. These oligonucleotides have been ~ound to modulate the expression of protein kinase C.
10 Palliation and therapeutic effect result.
.
,, BX~XGRO~ND OF T~ I ~ B~TIO~
The phosphoryla~:ion of proteins plays~;a key role. in the transduction of: extracellular ~ignals into the cell. ~he enzymes, called kinases, which effect such phosphorylations are 15 targetæ for the ac~ion of growth factors, hormones, and other agents involved in cellular~ metabolism, proliferation and di~ferentiation. O~e o~ the major signal transduction pathways , .
::~ involves ~h~ enzyme protein kinase C (PKC), which is known to have a critical~ influence on cell prolifera~ion and 20 diflferen~iatIon. /PKC is activated by:diacylglyc~rolsl(DAG~s), . which are:m~tabolites released in signaI transduction.
Interest in PKC:was s~imulated by the finding that PKC
is the maj~r, and perhaps only, cellular ~eceptor through which `: a ~lass o~ tumor-p~omoting agents called phoxbol esters exert Z:5~: their pleiotropic effec~s; on~cells. Gescher et~al., Anti~
: Cancer~ ~Drug Desi~n 1989~, 4, 93~-1050 Phorb~ls~capable of tum~r production~ can mimic the effect of DAG in activa~ing PKC, :, ~ : , :
W093/19203 P~T~US93/02213 2 1 3 2 09 l _ 2 - ! r suggesting that these tumor promotors act through PKC and that activation of this enzyme is at least partially responsible for the resul~ing tumorigenesis. Parker et al., Science 1986, 233, 853-866.
Experimental evidence indicates that PKC plays a role in growth control in colon cancer. It is believed that specific bacteria in the intestlnal tract convert lipids to DAG, thus activating PKC and altering cell:proliferation. This may explain the correlation between high dietary fat and colon cancer;. Weinstein, Cancer Res4 ~Suppl.) 1991, 51, 5080s~5085s.
It has also been demonstrated that a greater proportion of the PKC in th~ colonic mucosa of patients with colorectal cancer is _ in an activated state compared to that of patients without cancer. Sakanoue et al., Int . J. Can er 1991, 48, 803-806.
Increased tumorigenicity is also correlated with overexprèssion of PKC in cultured cells inoculated into nude . .
mice.~ A mutant ~orm of~ PXC induces highly malignant tumor cells with increased metastatic potential. Sphingoslne and related~ inhibitors of PKC activity have been ~hown to inhibit 20; tumor cell growth and radiation-induced transformation in vivo.
Endo et al., Cancer~Research 1991, 51, 1613-1618; Borek et al., P~oc. Natl. Acad. Scl. 1991, 88, 1953-1957. A number of experimental or clinically useful anti-cancer drugs show ~modulatory effects~on~PKC. Therefore,~inhibitors of PKC may be 25~ important cancer-preventive or therapeutic agents. PKC has been suggested as~a~plausible tar~et for~more rational design of con~entional anti-cancer drugs. Gescher, A. an~ Dale, I.L., :
Anti-Cancer Drug Design~ 198'~, ~, 93-105.
xperiments also indicate tha~ PKC plays an important 3G ~role~ in the pathophysiology of hyperproliferati~e skin ` disorders such~as~psoriasis~and skin cancer. ~Psoriasis is characterized~by~inflamma~ion,~ hyperproliferation ~of ~the epider~is~ nd~decreased~ dif~feréntiation~of~cells.~ Various studies indicate~a~role for PKC in causing~these symptoms. PKC
35~ stimulation in~çultured~keratinocytes can be shown ~o cause hyperproli~feration.~ ~Inflammation can be induced by phorbol esters ~and~ is regulated~by~P~C. DAG is implicated in ~he W093/19203 213 2 0 9 ~ PCT/US93/02213
- 3 -involvement of PKC in dermatological diseases, and is formed to an increased extent in psoriatic lesions.
Inhibitors of PKC ha~e been shown to have both antiproli~erative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis. Hegemann, L. and G. Mahrle, Pharmacoloqy_o~ the Skin, Ho Mukhtar, Ed., CRC ~?ress, ~oca Raton, FL, 1992, p.
10 357-368.
PKC is not a single enzyme, but a family of enzymes.
At the present time at least seven isoforms (isozymes) of PKC
have been identified: isoforms ~, B, and ~ have been purified to homogeneity, and iso~orms ~ and ~ have been identified 15 by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishiæuka, Nature 1988, 334, 66l-665 for review) and may serve different physiological functions. For example, PKC-~ seems to be expressed only in the central nerYous system. PKC-a and -B are expressed in most 20 tissues, but have different patterns of expression in different cell types. For example, both PKC-~ and PKC-B are expressed in, and have been purified from, hu~an epidermis. While PKC-~has been detected mainly~in keratinocytes of the basal layers of the epidermis, PKC-B is found mainly in the middle layers of ~5 the epidermis and Langerhans cells. PKC-~ has been found pre~ominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain. Osada et al., JO Biol.
30 Ch~m.;l990,i265/ 22434-22440. ~hile the PKC isbzymeis listed here are preferred for targeting by the present invention, ~ othèr isozymas o~ PKC are also~ co~prehended by the present invention.
:
:~ It is prese~tly beliPved that different PKC isozymes may 35 ~e involved in variou~ disea~e processes depending on the organ ~ ~ or tissue in which they are expressed~ For example, in :~ psoriatic lesions there is an alteration in the ratio between
Inhibitors of PKC ha~e been shown to have both antiproli~erative and antiinflammatory effects in vitro. Some antipsoriasis drugs, such as cyclosporine A and anthralin, have been shown to inhibit PKC. Inhibition of PKC has been suggested as a therapeutic approach to the treatment of psoriasis. Hegemann, L. and G. Mahrle, Pharmacoloqy_o~ the Skin, Ho Mukhtar, Ed., CRC ~?ress, ~oca Raton, FL, 1992, p.
10 357-368.
PKC is not a single enzyme, but a family of enzymes.
At the present time at least seven isoforms (isozymes) of PKC
have been identified: isoforms ~, B, and ~ have been purified to homogeneity, and iso~orms ~ and ~ have been identified 15 by molecular cloning. These isozymes have distinct patterns of tissue and organ localization (see Nishiæuka, Nature 1988, 334, 66l-665 for review) and may serve different physiological functions. For example, PKC-~ seems to be expressed only in the central nerYous system. PKC-a and -B are expressed in most 20 tissues, but have different patterns of expression in different cell types. For example, both PKC-~ and PKC-B are expressed in, and have been purified from, hu~an epidermis. While PKC-~has been detected mainly~in keratinocytes of the basal layers of the epidermis, PKC-B is found mainly in the middle layers of ~5 the epidermis and Langerhans cells. PKC-~ has been found pre~ominantly in the skin and lungs, with levels of expression much higher in these tissues than in the brain. This is in contrast to other members of the PKC family which tend to be most abundantly expressed in the brain. Osada et al., JO Biol.
30 Ch~m.;l990,i265/ 22434-22440. ~hile the PKC isbzymeis listed here are preferred for targeting by the present invention, ~ othèr isozymas o~ PKC are also~ co~prehended by the present invention.
:
:~ It is prese~tly beliPved that different PKC isozymes may 35 ~e involved in variou~ disea~e processes depending on the organ ~ ~ or tissue in which they are expressed~ For example, in :~ psoriatic lesions there is an alteration in the ratio between
- 4 PKC-~ and PKC-B, with preferential loss o~ ~XC-B compared to normal skin (Hegemann, ~. and G. Mahrle, PharmacoloqY of the Skin, H. Mukhtar, Ed., CRC Press, Boca Raton, FL, 1992, p. 357-, 368.
Although numerous compounds have been identified as PKC
inhibitors (see Hidaka and Hagiwara, Trends in Pharm . Sci .
19~7, 8, 162-164 for review), none has been found which ;;
lnhibits PKC specifically. While the quinoline sulfonamide derivatives such~ as ~-(5-isoquinolinesulfonyl)-Z-10 methylpiperazine (~I~7)~ inhibit PKC at micromolar concentrations, they e~hibit simiIar enzyme inhibition kinetics ~-for; PKC and the cAMP-dependent ~and cGMP-dependent protein kinases. Staurosporine~, an alkaloid product of Streptomyces ;
sp., and its analogs, are the most potent in ~itro jinhibitors of PKC identified to date. However, they exhibit only limited selectivity~ among di~fferent protein kinases. Gescher, Anti-Cancer~ Drug Design ~1989~, 4, 93-105.~ Thus there has been a ~ -ai1ure~of~others to inhibit PKC specifically. There is~also a deslre t~ inhibit specific PKC~lsozymes,~both as a research~;
20~ tool and as treatment for diseases which may be associated with ;
` particular isoz~mes.
~OF~E INVEN~ION~
It is a`~princlpal~ as~ct ~of the~invention to~ provide~
therapies~for~neoplastic,~hyperproliferative,~inflammatory~and~ ;
25 ~other~disea~e states~ass~o~ciated w1th protein kinase C ~
Another~ a5peC~ ;;of the invention lS to~provide selectlve therap1es~for~diseases associated wlth particular isozymes of protein k~naselC.~
It ~is~a~further~ aspect of ~the lnventlon to provide 30~antisense~01lg~onùc~1eotldes~whi~ch~are capable~o`f~modulating~the ;expression~of~pr~otein~kinase C. ~ ~ ;
Another~ aspsct~ of~the 1nvention~is~to provide antisense o~ go~nùcleotldes~whlch~ are~capable of selectively modulating the~expres~sion of partlCul~ar isozymes of~protein kinase`C
3`5~ ;Y~et anothér aspect~ s~to provlde means for dlagnosls of dlseases:~;a:ssociat~ed~Wlth proteln klnase~C~ ., A further aspect of the invention is to provide means for differential diagnosis of diseases associated with particular isozymes of protein kinase C.
A still further aspect of the invention is to provide
Although numerous compounds have been identified as PKC
inhibitors (see Hidaka and Hagiwara, Trends in Pharm . Sci .
19~7, 8, 162-164 for review), none has been found which ;;
lnhibits PKC specifically. While the quinoline sulfonamide derivatives such~ as ~-(5-isoquinolinesulfonyl)-Z-10 methylpiperazine (~I~7)~ inhibit PKC at micromolar concentrations, they e~hibit simiIar enzyme inhibition kinetics ~-for; PKC and the cAMP-dependent ~and cGMP-dependent protein kinases. Staurosporine~, an alkaloid product of Streptomyces ;
sp., and its analogs, are the most potent in ~itro jinhibitors of PKC identified to date. However, they exhibit only limited selectivity~ among di~fferent protein kinases. Gescher, Anti-Cancer~ Drug Design ~1989~, 4, 93-105.~ Thus there has been a ~ -ai1ure~of~others to inhibit PKC specifically. There is~also a deslre t~ inhibit specific PKC~lsozymes,~both as a research~;
20~ tool and as treatment for diseases which may be associated with ;
` particular isoz~mes.
~OF~E INVEN~ION~
It is a`~princlpal~ as~ct ~of the~invention to~ provide~
therapies~for~neoplastic,~hyperproliferative,~inflammatory~and~ ;
25 ~other~disea~e states~ass~o~ciated w1th protein kinase C ~
Another~ a5peC~ ;;of the invention lS to~provide selectlve therap1es~for~diseases associated wlth particular isozymes of protein k~naselC.~
It ~is~a~further~ aspect of ~the lnventlon to provide 30~antisense~01lg~onùc~1eotldes~whi~ch~are capable~o`f~modulating~the ;expression~of~pr~otein~kinase C. ~ ~ ;
Another~ aspsct~ of~the 1nvention~is~to provide antisense o~ go~nùcleotldes~whlch~ are~capable of selectively modulating the~expres~sion of partlCul~ar isozymes of~protein kinase`C
3`5~ ;Y~et anothér aspect~ s~to provlde means for dlagnosls of dlseases:~;a:ssociat~ed~Wlth proteln klnase~C~ ., A further aspect of the invention is to provide means for differential diagnosis of diseases associated with particular isozymes of protein kinase C.
A still further aspect of the invention is to provide
5 research tools for the study of the effect~ of protein kinase C e~pression and diseases associated therewith.
An additional aspect of the invention is to provide research tools for the study of the effects of expression of particular isozymes of protein kinase C and diseases associated 10 therewith.
These and other aspects of this invention will become apparent from a review of the instant specification~
BRIEF DE~CRIPTION OF TH~ D~WI~G8 Figure l(a) and l(b) are graphical depictions of the 15 effects on PKC expression of antisense oligonucleotides hybridizable with PKC ~. Oligonucleotides are arranged ~y PKC
target region, 5' to 3'.:
Figure 2 is a line graph showing dose-dependent antisense oligo~ucleotide inhibition of PKC-~ protein synthesis, expressed as percent of control ~no oligonucleotide). ~ = ISIS
3520; ~ = ISIS 3522; ~ = ISIS 3527; v = ISIS 4985.
Figure 3 is a line graph showing the dose-dependent inhibition of PKC-~ protein synthesis by ISIS 4649, the 2'-0-methyl version of ISIS 3522 (both are phosphorothioates). ~ =
ISIS 463~; ~ = ISIS 4636; ~ = ISIS 464~; ~ = ISIS 4648.
Figure 4 is a bar ~raph showing decrease in levels of PKC-~ mRNA transcripts after antisense oligonucleotide treatment. The hatched bars represent the 8.5 kb transcript i and the white bàrs represent ~he 4.0 kb transcript.
Flgure 5 is : a line graph showing antisense oligonucleotide inhibition of A549 cell proliferation. ~ =
4559;~ = 49g5;~ = 352~; ~ a 3527 .
Figure 6 is a series of line graphs showing inhibition of PKC~ mRNA levels by 2'-0-methyl phosphorothioate oligonucleotides ha~ing 8-deoxy gaps compared to full-deoxy phosphorothioate oligonucleotides having the same sequence.
: ~ :
WO 93/19203 PCr/US93/02213 F1~2 QA shows data for SEQ ID NO: 2 (5357 is gapped oligonucleotide, 3521 is full deoxy). Figure 6B shows data for SE~ ID NO: 3 (5352 is gapped oligonucleotide, 3522 is full d~oxy). Figure 6C shows data for SEQ ID NO: 5 ~5240 is gapped oligonucleotide, 3527 is ~ull deoxy).
Figure 7 is a line graph showing the relationship between d~oxy gap length (in a 2'-0-methyl oligonucleotide) and reduction of PKC~ mRNA levels by antisense oligonucleotides having SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5.
10 8~MMA~Y OF TEB INVBNTION
In accordance ~ with the pre~ent in~ention, ~ oligonucleotides are provided that are specifically hybridizable with DNA or RNA deriving from the gene that encodes PKC. The oligonucleotide comprises nucleotide units 15~ sufficient in identity and~ number to effeot such specific hybridization. This~ relationship~is commonly denominated as "antisense". In one preferred embodiment, the oligonucleotides are~speoifically hybridizable with the translation initiation codon of the gene,~ and~preferably comprise a sequence CAT. In 20 ~another preferred embodiment, the oligonucleotides are ; ;;;specifically ~hybridizable with ~the 5'-untranslated or 3' ~untranslated regions of the~gene. ~ In yet another preferred embodiment, oligonucleotides are provided that are specifically hybridizable with~DNA~or~mRNA encoding a particular PKC isozyme 25~ or~a part~icular set~of~PRC~isozym~s. Such oligonucleotides are co m eniently~and~desirably; presented in a pharmaceutically accep~able~carri~er. ~
In accordancé with other preferred embodiments, the gonucleotlde~s~are formulated~such that at lèast one of the linking~groups~between nucleotide units of the oligonucleotide comprises~ a~ sulfur-containing species such as a phosphorothioate moiety. In~still other preferred embodiments, the~oligonucleotides;~are~formulated such that at least one of the~nucleotides i ~m~dified~at the ~2/~position of the sugar.
35~5~om~e~examples of~such~preferred~modifications are ~'-O-alkyl arld~2~'-f;luoro modiflca:t;ions.: ~
7 ~ 9 ~1 ;
WO93~19203 PCT/U~93/02213 . 7 Other aspects of the invention are direc~ed to methods for modulating the expression of ~XC or of a particular PKC
i50zyme or set of isozymes in rells or tissues. Additional aspects of the invention are directed ~o methods of detection in cells or tissues of the DNA or RNA that encodes PKC and specific detection in cells or tissues of RNA or DNA that encodes particular PK~ isozymes~ Such methods comprise contacting cells or tissue~ suspected of containing said gene with oligonucleotides in accordance with the in~ention in order 10 to interfere with the effect of or to detect said ~NA or D~A.
Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having - a disease associated with PKC or one of its isozymes. Such methods comprise contacting the animal or cells or tissues or 15 a bodily fluid from the animal with oligonucleotides in accordance with the invention in order to modulate the e~pression of PK~, to treat conditions associated with PKC, or to effsct a diagnosis thereof.
DE~AI~D D~8CRX~TION OF ~ NTIO~
Antisense oliyonucleotides hold great promise as therapeutic agents for the treatment of many human diseases.
O1igonucleotidès specifically bind ~hybridize) to the complementary sequence of DNA, pre-~NA or mature mRNA, as defined by Watson-Crick base pairing, interfering with the f low of genetic information from DNA to protein. The properties of antisense oligonucIeotides which make them specific for their target sequence also make them extraordinarily versatile.
~ecause antisense oligonucleotides are long chains of monomeric units, they may be readily synthesized for any targe~ RNA
sequence. Numerous recent studies have documented the utility of anti~ense oligonucleotides as biochemical tools for studying target proteins. Rothenberg et al., J. Natl. Cancer Inst.
: l98g, 8l! l539-l5~4; Zon, G., PharmacPutlcal Res. 19~, 5, 53g-: 549. Because of recent advances in oligonucleotide chemistry and syn~hPsis of nuclease resistant oligonucleotides which exhibit enhanced cell~uptake, it is now possible to consider ,.
W093/192~ PCT/US93/0~213 2 ~3~ 09 ~ - 8 - ~ t~
the use of antisense oligonucleotides as a novel form of therapeutics.
Antisense oligonucleotides offer an ideal solution to the problems ~ncountered in prior art approache~. They can be 5 designed t~ selectively inhibit a given isoz~me or particular set of i60zymes, or to inhibi`t all mem~ers of a given family of isozymQ~. They avoid non-specific mechanisms such as free radical ~cavenging. ~ complete unaerstanding of enzyme mechanism is not needed to design specific inhibitors.
Current agents which modulate the activity or metabolism of protein kinase C exhibit many unacceptable side effects due to their lack of specificity, or they exhibit only limited - effectiveness in inhibiting the enzyme. The instant invention circumvents problems encountered by prior workers by modulating 15 the production of the enzyme, rather than inhibiting the enzyme directly, to achieve the therapeutic effect. In the instant invention, the oligonucleotide is designed to bind directly to mRNA or to a g~ne, ultimately modulating the amount of PXC
: protein made from the gene.
In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring ba~es and pentofuranosyl groups joined by native phosphodiester bonds. This te ~ effectiv~ly refers to naturally occurring species or ~yn~hetic species formed from 25 naturally occurring subunits or their close homologs. The term "oligonucleotidel' may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages.
" j , I ~
; 30 Exemplary among these are the phosphorothioates and other ~u1fur-containing species such as phosphorodithioates whi~h are known for use in the art. In accor~ance with some preferred ~ ~-embodimen~s, at~least one of the phosphodiester bonds of the : ~ : oligonucleotide have been substi~uted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells wher the RN~ or DN~ whose a~tivity to be modulated is located. It is preferred that such ..
~1~2`~
W093/l9203 PCT/US93/02213 _ g _ substitutions comprise phosphorothioate bonds, m thyl phosphonate bonds, ~hor~ chain alkyl or cycloalkyl s~ructures, or heteroatom-sub~titu~ed short chain alkyl ~tructures. Most preferred are those with CH2-NH-O-CH2, CH2-M~CH3)~O-CH2, CH2-0-5 N(CH3)-~H2, CH2-N(CH3)-N(CH3)-CH2 and O-N(CH3~-CH2-CHz back~ones (where phosphodiester is O-P-O-CH2)~ Also preferred are oligonucleotides having morpholino backbone structures.
Summerton, J.E. and Weller, D.D., U.S. Patent No: 5,034,506.
In other pre~erred embodiments, such as the protein-nucleic 10 acid (PN~) backbone, the phosphodiester backbone of the oligonucleotide may ~e replaced with a poly~mide backbone, the ba~es being:bound directly or indirectly to the aza nitrogen - atoms of the polyamide backbone. P.E. Nielsen, M. Egholm, RuH.
Berg, O. Buchardt, Science ls~1, 254, 1497. In accordance with 15 other preferred embodiments, the phosphodiester bonds are substituted with other structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enan~iomerically specific. Persons of ordinary sXill in the ar~ will be able to select other linkages 20 for use in practice of the invention.
Oligonucleotides may ~lso include species which include at least one modified base form. Thus, purines and pyrimidines other than those normally found in nature may be so employed.
Similarly, modifications on the pentofuranosyl portion of the 25 nucleotide subunits may also be effected, as long as the ~es~ential tenets of this in~ention axe adhered to. Examples of : such modifications are 2'-O alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of modifications at the 2' :position of sugar moieties which are useful in the present 0 invention are OH, SH,~SCH3, F,:OCH3, OCN, O(CHz~nNH2 or O(CH2)nCH
where n is:~rom l:to: about 10,~ and other substituents having ; ~ similar properties. Sugar mimetics such as cyclobutyls or :~ ~ :o~her carbocycles may also~ be used in p~ace of the :: pentofurano~yl group.~Chimeric oligonucleotides having two or ::35 more chemically:distinct regions are also useful in the present invention. Specific examples of~such chimeric oligonucleotides ~;are those which are modified at the 2' position except for a ; ~
~ :
, WO93/19203 PCT/US93~221~
9~ - 10 ~
"deoxy gapl' of one or more devxynucleotides. The "deoxy gap"
may be cen~ered in the molecule or may be located at or near either end. Oligonucleotides with regions of distinct backbone chemistries are also examples of chimeric oligonucleotides.
Such oligonucleotides are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines~, but having one or more differences from natur~l structure. All such oligonucleotides are comprehended by this invention so long as 10 they function effectiYely to hybridizP with the PKC RNA. The oligonucleotides in accordance with this invention preferably comprise from about S to a~out 50 nucleotide units. It is more - preferred that such oligonucleotides comprise from about 8 to 30 nucleotide units, and still more preferred to have from about 12 to 25 nucleotide units. As will be apprec`iated, a nucleotide unit is a base-sugar combination suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for s~ch synthesis may also be employed; the a~tual synthe~is of the oligonucleotides is well 25 within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information t~ encode a pro~ein uslng ~he three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the ~'-un~ranslated regiQn, ~he 3~-untranslated ~egion, ~he 5' cap region and intronlexon junction ribonucleotides. Thus, oligonucleotides 35 may be formula~ed in accordance with this inventiQn which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides~ In preferred .
W093/192~3 2 i 3 2 ~ 9 ~ PCT/U~93/02213 j, ~
embodiments, the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation 5 ite, a 5' cap region, an intron/exon junction, coding sequence~ or sequences in the 5'- or 3'-untranslated region.
The oligonu~leotides of this invention are designed to be hybridizable with messenger R~A derived from the PKC gene.
Such hybridization, when accomplished, interfer s with the normal roles of the messenger RNA to cause a modulation of its function in the cell. The functions of messenger RNA to be interfered wi~h incl~de all vital functions such as transcription of the RNA from DNA, translocation of the RNA to the site for protein translation, actual translation of protein from the ~NA, splicing of the RN~ to yield one or more mRNA
specie~, and possibly even independent catalytic activity which 15 may be engaged in by the RNA. The overall effect of such interference with the RNA function is to modulate expression of the PKC gene.
The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research 20 reagen~s and kit~. Since the oligonucleotides of this invention hybridi~e ~o the PKC gene and its mRNA, sandwich and other assays can easily be constructed to exploit this fact.
Furthermore, since the oligonucleotides of this invention hybridize specifically to particular isozymes of the PKC mRNA, 25 such as~ays can be devised for screening of cells and tissues : ~r particular PKC isozymes. Such assays can be utilized for diagnosis of diseases associated with various P~C forms~
Provision of means for detecting hybridi~ation of oligonucleQtide with the PKC gene can routinely be 30 ~accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for d~tecting the presence or absence of PXC may also be prepar~
For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention . Oligonucleotides may be formulated in a pharmaceutical composition, whi~h may include carriers, .
WO93/19~03 PCT/US93/02213 2~3~ 09 - 12 ~ `
thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide.
Pharmac~utical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory 5 agents, ~nesthetics, and the like in addition to oligonucleotides. `
The pharmaceutical aomposition may be administered in a num~er of ways depending on whether local or systemic treatment i~ de~ired, and on the area to be treated. Administration may lO be done topically (including ophthalmically, vaginally, rectally, intranasally) r orally, by inhalation~ or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injection.
Formulations for topical administration may include 15 oin~ments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.; Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also b useful.
Compositions for oral administration include powders or 20 granules, suspensions or solutions in water or non-aqueous media, capsules, sache~s, or tablets. Thickeners, flavorings, diIuents, emulsifiers, dispersing aids or binders may ~e desirable.
Formulations for parenteral administration may include sterile aqueous solu~ions which may also contain buffers, diluents and other suitable addi~ives.
Dosing is dependent on severity and responsiveness of the condition to b~ treated, but will normally be one or more doses per day, with course of treatment lasting from several days to se~eral months or until a cure:is ef~ected or a diminu~ion of disease state is achieved. Persons of ordinary skill can easily determine: optimum dosayes, dosing methodologies and repetition rates, : The following:examples illustrate ~he presen~ invention 35 and are not intended to limit the same.
~'~
;:~
WO93/19203 2 1 3 ~ O 9 ~ ~CT/US93/02213 ~A~P~8 B~plo 1 Ol~go~ucl~oti~o ~y~th~s~
Unmodified DNA oligcnucleotide6 are synthesized on an automated DNA synthesizer (~pplied Biosystems model 380B) using 5 standard phosphoramidite chem~ætry with oxidation by iodine.
B-Cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle is replaced ~y a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,~-dioxide in lQ acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step is increased to 68 seconds and is followed b~ the capping step.
:After cleavage from :the controlled pore glass cvlumn (Applied Biosystems) and deblocking in concentrated ammonium 15 hydroxide at S5C for 18 hours, the oligonucleotides are purified by p~ecipitation twice out of 0.5 M NaCl with 2.5 volumes: ethanol. Analytical gel electrophoresis is accomplished in 20~ acrylamide, 8 ~ urea, 45 m~ Tris-borate buffer, pH 7Ø
20 Ex~ple 2 C~ll culture ~D~ traat~t ~ith phorbol esterY ~d oligo~uclo~tid~ targote~ to P~C-~ :
: PKC protein hal~-lives have b~en reported to vary from : 6.7 hours to over 24 hours. Young et al~, Biochem . J. 1~87, : : 244, 775-779; Ballester et ~aI~, J. Biol. ~hemO S9B5, 260, 1519~-15199. These long half-lives make inhibiting steady-sta~e levels of PKC-~ an unwieldy approach when screening antisense oligonu~lèotides, due to the long incu~ation times which would be re~uired. The ability of phorbol esters to re~ersibly l~wer intracellular levels of PKC has therefore been 30 exploited. Traatmen~ of cells with phorbol esters causes an -~.
initial activation of kina~e acti~ity, followed by a down-regulation of PXC. For~ PKc-~ this down-regulation has been : ~ shown to be a direct consequence of an increased rate of ~ proteolysis of the kinase~with no apparent change in synthetic : ~ ` 35 rate.
' ~
WO93/19~03 PCr/US93/02213 ~3~ 4 - 14 - _ :
It was initially determined that in human lung carcinoma (A54g) cells, treatment with the phorbol ester 12,13-dibutyrate (PDBu), using a modification of the method of Xrug et al., J.
Bio7. Chem. 1987, 2~2, 11852-11856, did indeed lower cellular levels of PKC-~, without affecting PKC-a mRNA levels, and that this e~fect wa~ reversible. The ba~is n~ the assay to screen for potency of oligonucleotides targeting PKC-~ i5 to initially lower PK~-~ protein level~ by chronic treatment with PDBu, remove PDBu by extensively washing the cells (hence allowing 10 the cells to synthesi2e fresh PKC-~ protein), and incubate the cells with oligonucleotides intended to-inhibit the resynthesis of new PKC-u protein.
- A549 cells (obtained from the American Type Culture Collection7 Bethesda MD) were grown to confluence in 6-well 15 plates ~Falcon Labware, Lincoln Park, NJ) in Dùlbecco's modified Eagle's medium (DM~) containing 1 g glucose/liter and 10% fetal calf serum (FCS, IrYine Scientific, Santa Ana, CA).
Cells were treated with S00 nM PDBu ~Sigma Chem. Co., St.
Louis, M0~ for 12-16 hours ~overnight). Cells were th~n washed 20 three times in DME at 37, and 1 ml DMA containing 20 ~1 DOT~A
(Lipofectin reagent, BRL, Bethesda, MD) was added.
Phosphorothioate oligonucleotides were added to a concentration of 1 ~ and the cells were incubated for a further 4 hours at 37C~
Cel 15 were washed once in 3 ml D~E containing 0.1 mg/ml BSA and a further 2 ml DME containing 0.1 mg/ml BSA was added.
Phosphorothioate oligonucleotides (1 ~M) were added and the cells were incubated at 37C for 24 hours.
Cells were washed three times in phosphate-buffered saiine (P~S~ and cellular proteins were extracted in 1~0 sample buffer (60 mM Tris pH 6.8, 2~ SDS, 10% glycerol, 10 m~
dithiothreitol) and boiled for 5 minutes. Intracellular levels of PKC-~ protein were determined by immunoblotting~
The oligonucleotides tested in this assay are presented in Table 1. Sequence data are from the cDNA sequence published by Fir~enzeller et al., NUC1. Acids Res. 1990, 1~, 21B3; the sequence numbers given under the oligonucleotides are relative W093/1~203 2132~9~ PCr/US93/0~213 - 15 ~
tc the first residue to be sequenced on the cDNA, which is 28 residues upstream of the ATG start codon. ~ i TAB~E 1 OLIGONUCLEOTIDES TARGE~ED TO HUMAN PKC-~ -5(All a~e phosphorothioates) SEQ I~ SEQUENCE TARGET
1 CCC CAA CCA CCT CTT GCT CC 5' Untranslated 2 GTT CTC GCT GG~ GAG TTT CA 3' Untranslated l02063 2044 3 AA~ ACG TCA GCC ATG GTC CC Translation init. codon ~-:
4 GGA TTC ACT TCC ACT GCG GG 3' Untranslated 2l09 2090 GAG ACC CTG AAC AGT TGA TC 3' Untranslated : 2211 2192
An additional aspect of the invention is to provide research tools for the study of the effects of expression of particular isozymes of protein kinase C and diseases associated 10 therewith.
These and other aspects of this invention will become apparent from a review of the instant specification~
BRIEF DE~CRIPTION OF TH~ D~WI~G8 Figure l(a) and l(b) are graphical depictions of the 15 effects on PKC expression of antisense oligonucleotides hybridizable with PKC ~. Oligonucleotides are arranged ~y PKC
target region, 5' to 3'.:
Figure 2 is a line graph showing dose-dependent antisense oligo~ucleotide inhibition of PKC-~ protein synthesis, expressed as percent of control ~no oligonucleotide). ~ = ISIS
3520; ~ = ISIS 3522; ~ = ISIS 3527; v = ISIS 4985.
Figure 3 is a line graph showing the dose-dependent inhibition of PKC-~ protein synthesis by ISIS 4649, the 2'-0-methyl version of ISIS 3522 (both are phosphorothioates). ~ =
ISIS 463~; ~ = ISIS 4636; ~ = ISIS 464~; ~ = ISIS 4648.
Figure 4 is a bar ~raph showing decrease in levels of PKC-~ mRNA transcripts after antisense oligonucleotide treatment. The hatched bars represent the 8.5 kb transcript i and the white bàrs represent ~he 4.0 kb transcript.
Flgure 5 is : a line graph showing antisense oligonucleotide inhibition of A549 cell proliferation. ~ =
4559;~ = 49g5;~ = 352~; ~ a 3527 .
Figure 6 is a series of line graphs showing inhibition of PKC~ mRNA levels by 2'-0-methyl phosphorothioate oligonucleotides ha~ing 8-deoxy gaps compared to full-deoxy phosphorothioate oligonucleotides having the same sequence.
: ~ :
WO 93/19203 PCr/US93/02213 F1~2 QA shows data for SEQ ID NO: 2 (5357 is gapped oligonucleotide, 3521 is full deoxy). Figure 6B shows data for SE~ ID NO: 3 (5352 is gapped oligonucleotide, 3522 is full d~oxy). Figure 6C shows data for SEQ ID NO: 5 ~5240 is gapped oligonucleotide, 3527 is ~ull deoxy).
Figure 7 is a line graph showing the relationship between d~oxy gap length (in a 2'-0-methyl oligonucleotide) and reduction of PKC~ mRNA levels by antisense oligonucleotides having SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5.
10 8~MMA~Y OF TEB INVBNTION
In accordance ~ with the pre~ent in~ention, ~ oligonucleotides are provided that are specifically hybridizable with DNA or RNA deriving from the gene that encodes PKC. The oligonucleotide comprises nucleotide units 15~ sufficient in identity and~ number to effeot such specific hybridization. This~ relationship~is commonly denominated as "antisense". In one preferred embodiment, the oligonucleotides are~speoifically hybridizable with the translation initiation codon of the gene,~ and~preferably comprise a sequence CAT. In 20 ~another preferred embodiment, the oligonucleotides are ; ;;;specifically ~hybridizable with ~the 5'-untranslated or 3' ~untranslated regions of the~gene. ~ In yet another preferred embodiment, oligonucleotides are provided that are specifically hybridizable with~DNA~or~mRNA encoding a particular PKC isozyme 25~ or~a part~icular set~of~PRC~isozym~s. Such oligonucleotides are co m eniently~and~desirably; presented in a pharmaceutically accep~able~carri~er. ~
In accordancé with other preferred embodiments, the gonucleotlde~s~are formulated~such that at lèast one of the linking~groups~between nucleotide units of the oligonucleotide comprises~ a~ sulfur-containing species such as a phosphorothioate moiety. In~still other preferred embodiments, the~oligonucleotides;~are~formulated such that at least one of the~nucleotides i ~m~dified~at the ~2/~position of the sugar.
35~5~om~e~examples of~such~preferred~modifications are ~'-O-alkyl arld~2~'-f;luoro modiflca:t;ions.: ~
7 ~ 9 ~1 ;
WO93~19203 PCT/U~93/02213 . 7 Other aspects of the invention are direc~ed to methods for modulating the expression of ~XC or of a particular PKC
i50zyme or set of isozymes in rells or tissues. Additional aspects of the invention are directed ~o methods of detection in cells or tissues of the DNA or RNA that encodes PKC and specific detection in cells or tissues of RNA or DNA that encodes particular PK~ isozymes~ Such methods comprise contacting cells or tissue~ suspected of containing said gene with oligonucleotides in accordance with the in~ention in order 10 to interfere with the effect of or to detect said ~NA or D~A.
Other aspects of the invention are directed to methods for diagnostics and therapeutics of animals suspected of having - a disease associated with PKC or one of its isozymes. Such methods comprise contacting the animal or cells or tissues or 15 a bodily fluid from the animal with oligonucleotides in accordance with the invention in order to modulate the e~pression of PK~, to treat conditions associated with PKC, or to effsct a diagnosis thereof.
DE~AI~D D~8CRX~TION OF ~ NTIO~
Antisense oliyonucleotides hold great promise as therapeutic agents for the treatment of many human diseases.
O1igonucleotidès specifically bind ~hybridize) to the complementary sequence of DNA, pre-~NA or mature mRNA, as defined by Watson-Crick base pairing, interfering with the f low of genetic information from DNA to protein. The properties of antisense oligonucIeotides which make them specific for their target sequence also make them extraordinarily versatile.
~ecause antisense oligonucleotides are long chains of monomeric units, they may be readily synthesized for any targe~ RNA
sequence. Numerous recent studies have documented the utility of anti~ense oligonucleotides as biochemical tools for studying target proteins. Rothenberg et al., J. Natl. Cancer Inst.
: l98g, 8l! l539-l5~4; Zon, G., PharmacPutlcal Res. 19~, 5, 53g-: 549. Because of recent advances in oligonucleotide chemistry and syn~hPsis of nuclease resistant oligonucleotides which exhibit enhanced cell~uptake, it is now possible to consider ,.
W093/192~ PCT/US93/0~213 2 ~3~ 09 ~ - 8 - ~ t~
the use of antisense oligonucleotides as a novel form of therapeutics.
Antisense oligonucleotides offer an ideal solution to the problems ~ncountered in prior art approache~. They can be 5 designed t~ selectively inhibit a given isoz~me or particular set of i60zymes, or to inhibi`t all mem~ers of a given family of isozymQ~. They avoid non-specific mechanisms such as free radical ~cavenging. ~ complete unaerstanding of enzyme mechanism is not needed to design specific inhibitors.
Current agents which modulate the activity or metabolism of protein kinase C exhibit many unacceptable side effects due to their lack of specificity, or they exhibit only limited - effectiveness in inhibiting the enzyme. The instant invention circumvents problems encountered by prior workers by modulating 15 the production of the enzyme, rather than inhibiting the enzyme directly, to achieve the therapeutic effect. In the instant invention, the oligonucleotide is designed to bind directly to mRNA or to a g~ne, ultimately modulating the amount of PXC
: protein made from the gene.
In the context of this invention, the term "oligonucleotide" refers to a polynucleotide formed from naturally occurring ba~es and pentofuranosyl groups joined by native phosphodiester bonds. This te ~ effectiv~ly refers to naturally occurring species or ~yn~hetic species formed from 25 naturally occurring subunits or their close homologs. The term "oligonucleotidel' may also refer to moieties which function similarly to naturally occurring oligonucleotides but which have non-naturally occurring portions. Thus, oligonucleotides may have altered sugar moieties or inter-sugar linkages.
" j , I ~
; 30 Exemplary among these are the phosphorothioates and other ~u1fur-containing species such as phosphorodithioates whi~h are known for use in the art. In accor~ance with some preferred ~ ~-embodimen~s, at~least one of the phosphodiester bonds of the : ~ : oligonucleotide have been substi~uted with a structure which functions to enhance the ability of the compositions to penetrate into the region of cells wher the RN~ or DN~ whose a~tivity to be modulated is located. It is preferred that such ..
~1~2`~
W093/l9203 PCT/US93/02213 _ g _ substitutions comprise phosphorothioate bonds, m thyl phosphonate bonds, ~hor~ chain alkyl or cycloalkyl s~ructures, or heteroatom-sub~titu~ed short chain alkyl ~tructures. Most preferred are those with CH2-NH-O-CH2, CH2-M~CH3)~O-CH2, CH2-0-5 N(CH3)-~H2, CH2-N(CH3)-N(CH3)-CH2 and O-N(CH3~-CH2-CHz back~ones (where phosphodiester is O-P-O-CH2)~ Also preferred are oligonucleotides having morpholino backbone structures.
Summerton, J.E. and Weller, D.D., U.S. Patent No: 5,034,506.
In other pre~erred embodiments, such as the protein-nucleic 10 acid (PN~) backbone, the phosphodiester backbone of the oligonucleotide may ~e replaced with a poly~mide backbone, the ba~es being:bound directly or indirectly to the aza nitrogen - atoms of the polyamide backbone. P.E. Nielsen, M. Egholm, RuH.
Berg, O. Buchardt, Science ls~1, 254, 1497. In accordance with 15 other preferred embodiments, the phosphodiester bonds are substituted with other structures which are, at once, substantially non-ionic and non-chiral, or with structures which are chiral and enan~iomerically specific. Persons of ordinary sXill in the ar~ will be able to select other linkages 20 for use in practice of the invention.
Oligonucleotides may ~lso include species which include at least one modified base form. Thus, purines and pyrimidines other than those normally found in nature may be so employed.
Similarly, modifications on the pentofuranosyl portion of the 25 nucleotide subunits may also be effected, as long as the ~es~ential tenets of this in~ention axe adhered to. Examples of : such modifications are 2'-O alkyl- and 2'-halogen-substituted nucleotides. Some specific examples of modifications at the 2' :position of sugar moieties which are useful in the present 0 invention are OH, SH,~SCH3, F,:OCH3, OCN, O(CHz~nNH2 or O(CH2)nCH
where n is:~rom l:to: about 10,~ and other substituents having ; ~ similar properties. Sugar mimetics such as cyclobutyls or :~ ~ :o~her carbocycles may also~ be used in p~ace of the :: pentofurano~yl group.~Chimeric oligonucleotides having two or ::35 more chemically:distinct regions are also useful in the present invention. Specific examples of~such chimeric oligonucleotides ~;are those which are modified at the 2' position except for a ; ~
~ :
, WO93/19203 PCT/US93~221~
9~ - 10 ~
"deoxy gapl' of one or more devxynucleotides. The "deoxy gap"
may be cen~ered in the molecule or may be located at or near either end. Oligonucleotides with regions of distinct backbone chemistries are also examples of chimeric oligonucleotides.
Such oligonucleotides are best described as being functionally interchangeable with natural oligonucleotides (or synthesized oligonucleotides along natural lines~, but having one or more differences from natur~l structure. All such oligonucleotides are comprehended by this invention so long as 10 they function effectiYely to hybridizP with the PKC RNA. The oligonucleotides in accordance with this invention preferably comprise from about S to a~out 50 nucleotide units. It is more - preferred that such oligonucleotides comprise from about 8 to 30 nucleotide units, and still more preferred to have from about 12 to 25 nucleotide units. As will be apprec`iated, a nucleotide unit is a base-sugar combination suitably bound to an adjacent nucleotide unit through phosphodiester or other bonds.
The oligonucleotides used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including Applied Biosystems. Any other means for s~ch synthesis may also be employed; the a~tual synthe~is of the oligonucleotides is well 25 within the talents of the routineer. It is also well known to use similar techniques to prepare other oligonucleotides such as the phosphorothioates and alkylated derivatives.
In accordance with this invention, persons of ordinary skill in the art will understand that messenger RNA includes not only the information t~ encode a pro~ein uslng ~he three letter genetic code, but also associated ribonucleotides which form a region known to such persons as the ~'-un~ranslated regiQn, ~he 3~-untranslated ~egion, ~he 5' cap region and intronlexon junction ribonucleotides. Thus, oligonucleotides 35 may be formula~ed in accordance with this inventiQn which are targeted wholly or in part to these associated ribonucleotides as well as to the informational ribonucleotides~ In preferred .
W093/192~3 2 i 3 2 ~ 9 ~ PCT/U~93/02213 j, ~
embodiments, the oligonucleotide is specifically hybridizable with a transcription initiation site, a translation initiation 5 ite, a 5' cap region, an intron/exon junction, coding sequence~ or sequences in the 5'- or 3'-untranslated region.
The oligonu~leotides of this invention are designed to be hybridizable with messenger R~A derived from the PKC gene.
Such hybridization, when accomplished, interfer s with the normal roles of the messenger RNA to cause a modulation of its function in the cell. The functions of messenger RNA to be interfered wi~h incl~de all vital functions such as transcription of the RNA from DNA, translocation of the RNA to the site for protein translation, actual translation of protein from the ~NA, splicing of the RN~ to yield one or more mRNA
specie~, and possibly even independent catalytic activity which 15 may be engaged in by the RNA. The overall effect of such interference with the RNA function is to modulate expression of the PKC gene.
The oligonucleotides of this invention can be used in diagnostics, therapeutics, prophylaxis, and as research 20 reagen~s and kit~. Since the oligonucleotides of this invention hybridi~e ~o the PKC gene and its mRNA, sandwich and other assays can easily be constructed to exploit this fact.
Furthermore, since the oligonucleotides of this invention hybridize specifically to particular isozymes of the PKC mRNA, 25 such as~ays can be devised for screening of cells and tissues : ~r particular PKC isozymes. Such assays can be utilized for diagnosis of diseases associated with various P~C forms~
Provision of means for detecting hybridi~ation of oligonucleQtide with the PKC gene can routinely be 30 ~accomplished. Such provision may include enzyme conjugation, radiolabelling or any other suitable detection systems. Kits for d~tecting the presence or absence of PXC may also be prepar~
For therapeutic or prophylactic treatment, oligonucleotides are administered in accordance with this invention . Oligonucleotides may be formulated in a pharmaceutical composition, whi~h may include carriers, .
WO93/19~03 PCT/US93/02213 2~3~ 09 - 12 ~ `
thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the oligonucleotide.
Pharmac~utical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory 5 agents, ~nesthetics, and the like in addition to oligonucleotides. `
The pharmaceutical aomposition may be administered in a num~er of ways depending on whether local or systemic treatment i~ de~ired, and on the area to be treated. Administration may lO be done topically (including ophthalmically, vaginally, rectally, intranasally) r orally, by inhalation~ or parenterally, for example by intravenous drip or subcutaneous, intraperitoneal or intramuscular injection.
Formulations for topical administration may include 15 oin~ments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.; Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms may also b useful.
Compositions for oral administration include powders or 20 granules, suspensions or solutions in water or non-aqueous media, capsules, sache~s, or tablets. Thickeners, flavorings, diIuents, emulsifiers, dispersing aids or binders may ~e desirable.
Formulations for parenteral administration may include sterile aqueous solu~ions which may also contain buffers, diluents and other suitable addi~ives.
Dosing is dependent on severity and responsiveness of the condition to b~ treated, but will normally be one or more doses per day, with course of treatment lasting from several days to se~eral months or until a cure:is ef~ected or a diminu~ion of disease state is achieved. Persons of ordinary skill can easily determine: optimum dosayes, dosing methodologies and repetition rates, : The following:examples illustrate ~he presen~ invention 35 and are not intended to limit the same.
~'~
;:~
WO93/19203 2 1 3 ~ O 9 ~ ~CT/US93/02213 ~A~P~8 B~plo 1 Ol~go~ucl~oti~o ~y~th~s~
Unmodified DNA oligcnucleotide6 are synthesized on an automated DNA synthesizer (~pplied Biosystems model 380B) using 5 standard phosphoramidite chem~ætry with oxidation by iodine.
B-Cyanoethyldiisopropyl-phosphoramidites are purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle is replaced ~y a 0.2 M solution of 3H-1,2-benzodithiole-3-one 1,~-dioxide in lQ acetonitrile for the stepwise thiation of the phosphite linkages. The thiation cycle wait step is increased to 68 seconds and is followed b~ the capping step.
:After cleavage from :the controlled pore glass cvlumn (Applied Biosystems) and deblocking in concentrated ammonium 15 hydroxide at S5C for 18 hours, the oligonucleotides are purified by p~ecipitation twice out of 0.5 M NaCl with 2.5 volumes: ethanol. Analytical gel electrophoresis is accomplished in 20~ acrylamide, 8 ~ urea, 45 m~ Tris-borate buffer, pH 7Ø
20 Ex~ple 2 C~ll culture ~D~ traat~t ~ith phorbol esterY ~d oligo~uclo~tid~ targote~ to P~C-~ :
: PKC protein hal~-lives have b~en reported to vary from : 6.7 hours to over 24 hours. Young et al~, Biochem . J. 1~87, : : 244, 775-779; Ballester et ~aI~, J. Biol. ~hemO S9B5, 260, 1519~-15199. These long half-lives make inhibiting steady-sta~e levels of PKC-~ an unwieldy approach when screening antisense oligonu~lèotides, due to the long incu~ation times which would be re~uired. The ability of phorbol esters to re~ersibly l~wer intracellular levels of PKC has therefore been 30 exploited. Traatmen~ of cells with phorbol esters causes an -~.
initial activation of kina~e acti~ity, followed by a down-regulation of PXC. For~ PKc-~ this down-regulation has been : ~ shown to be a direct consequence of an increased rate of ~ proteolysis of the kinase~with no apparent change in synthetic : ~ ` 35 rate.
' ~
WO93/19~03 PCr/US93/02213 ~3~ 4 - 14 - _ :
It was initially determined that in human lung carcinoma (A54g) cells, treatment with the phorbol ester 12,13-dibutyrate (PDBu), using a modification of the method of Xrug et al., J.
Bio7. Chem. 1987, 2~2, 11852-11856, did indeed lower cellular levels of PKC-~, without affecting PKC-a mRNA levels, and that this e~fect wa~ reversible. The ba~is n~ the assay to screen for potency of oligonucleotides targeting PKC-~ i5 to initially lower PK~-~ protein level~ by chronic treatment with PDBu, remove PDBu by extensively washing the cells (hence allowing 10 the cells to synthesi2e fresh PKC-~ protein), and incubate the cells with oligonucleotides intended to-inhibit the resynthesis of new PKC-u protein.
- A549 cells (obtained from the American Type Culture Collection7 Bethesda MD) were grown to confluence in 6-well 15 plates ~Falcon Labware, Lincoln Park, NJ) in Dùlbecco's modified Eagle's medium (DM~) containing 1 g glucose/liter and 10% fetal calf serum (FCS, IrYine Scientific, Santa Ana, CA).
Cells were treated with S00 nM PDBu ~Sigma Chem. Co., St.
Louis, M0~ for 12-16 hours ~overnight). Cells were th~n washed 20 three times in DME at 37, and 1 ml DMA containing 20 ~1 DOT~A
(Lipofectin reagent, BRL, Bethesda, MD) was added.
Phosphorothioate oligonucleotides were added to a concentration of 1 ~ and the cells were incubated for a further 4 hours at 37C~
Cel 15 were washed once in 3 ml D~E containing 0.1 mg/ml BSA and a further 2 ml DME containing 0.1 mg/ml BSA was added.
Phosphorothioate oligonucleotides (1 ~M) were added and the cells were incubated at 37C for 24 hours.
Cells were washed three times in phosphate-buffered saiine (P~S~ and cellular proteins were extracted in 1~0 sample buffer (60 mM Tris pH 6.8, 2~ SDS, 10% glycerol, 10 m~
dithiothreitol) and boiled for 5 minutes. Intracellular levels of PKC-~ protein were determined by immunoblotting~
The oligonucleotides tested in this assay are presented in Table 1. Sequence data are from the cDNA sequence published by Fir~enzeller et al., NUC1. Acids Res. 1990, 1~, 21B3; the sequence numbers given under the oligonucleotides are relative W093/1~203 2132~9~ PCr/US93/0~213 - 15 ~
tc the first residue to be sequenced on the cDNA, which is 28 residues upstream of the ATG start codon. ~ i TAB~E 1 OLIGONUCLEOTIDES TARGE~ED TO HUMAN PKC-~ -5(All a~e phosphorothioates) SEQ I~ SEQUENCE TARGET
1 CCC CAA CCA CCT CTT GCT CC 5' Untranslated 2 GTT CTC GCT GG~ GAG TTT CA 3' Untranslated l02063 2044 3 AA~ ACG TCA GCC ATG GTC CC Translation init. codon ~-:
4 GGA TTC ACT TCC ACT GCG GG 3' Untranslated 2l09 2090 GAG ACC CTG AAC AGT TGA TC 3' Untranslated : 2211 2192
6 CCC GGG AA~ ACG TCA GCC AT Translation init. codon 47 28 :
7 CTG CCT CAG CGC CCC TTT GC Internal (Cl) domain --:
0 1 1 0 9 1 ~ `
~ .
0 1 1 0 9 1 ~ `
~ .
8 AGT CGG TGC AGT GGC T GG AG Internal (Cl) domain ~:
: 193 : 174 g ~CA GAG GCT GGG GAC ATT GA Internal (Cl) domain . -~
: 480 461 25~:10 GGG CTG GGG ~GG~TGT TTG TT 3' Untranslated 2080 ~ ~ 2061 ~ :
~` ` ll . CAC TGC GGG G~G GGC TG~ GG 3t Untranslated :: 20~ 20~9 : ~:
l2 ~ AGC CGT GGC CTT ~ ATT TT j 3' Untranslated 301 i 2137 ~ 2118 :
~:~:: 13~ ~TT TTC AG~ CCT~CCA TAT ~G 3' Untranslated 2168~ 2149 l4 AAG AGA GAG A~C:CTG~AAC AG 3' Untranslated .
:: 2217: ~ 2198 35 15 ~GAT AAT GTT CTT G&~ TGT ~A 3' Untranslated 2235 ~ :~2216 ;
WV93/19~03 Pcr/uss3/o22l3 '1~3~ 16 ~
16 ATG GGG TGC ACA AAC TGG GG Internal (C3) domain 17 GTC AGC CAT GGT CCC CCC CC Translation init. codon 18 CGC CGT GG~ GTC GTT GCC CG Internal ~Vl) domain 63 44 -`
i9 TCA AAT GGA GGC TGC CCG GC Internal (C3) domain .
TGG AAT C~G ACA CAA GCC GT 3' Untranslated 21~1 2132 .
E~a~ple 3 I~u~oblot ~ay for P~C ~xpr~3~ion - Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. The resolved proteins were transferred to I~mobilon-P
membrane (Millipore, Bedford MA) by electrophoretic transfer }5 and the membrane was blocked for 60 minutes in TBS (Tris-HCl pH
7.4, 15Q mM NaC1) contain~;.ng 5% nonfat milk. The membrane was then incubated for 16 hours at 4C with monoclonal antibodies raised against PKC-~ (UBX, Lake Placid NY) diluted to 0.2 ~gjml in TBS containing 0.2$ nonfat milk. This was followed by three 20 washes in TBS plus 0.2% nonfat milk. The membrane was ~hen incubated ~or one hour with l2sI-labelled goat anti-mouse secondary antibody (ICN Radiochemicals, Irvine CA). Membranes were then washed extensively in TBS plus 0.2% nonfat milk.
: Bands were ~isualized and quantit~ted using a Phosphorimager (:Molecular Dynamics, Sunnyvale, CA). PKC-~ appears as a single band with a molecular weight of 80 kD.
Each oligonucleotide was tested three times, in trîplicate, and the results of the experiments were normalized a~ainst percentage of protein present as compared to cells ~0 which were not~treated with oligonucleotide (Figure 1). The j~.
:~~ive most e~fective oligonucleotides target the AUG start codon ~:
and regions slightly upstream and downstream from it ~oligos 1, --3;, 17, 7, 6)o The next most effective oligonucleotides are targeted toward the 3' unt~anslated region of the RN~ ~oligos 2, 5j 14).
.,:
:
W093/~9203 2 ~ ~ 2 3 9 ~ PCT/US93/02213 ~ampl~ ~ Ot~r i~o~y~3 of P~C
It was found that the most ef~ective sequences for antisense targeting of h~man PKC~ are those surrounding the transl~tion initiation codon and the 3' untranslated region.
It is believed that these sequences would also be effective targets for oligonucleotides directed against other isozymes of PKC. The other isozymes of human PKC for which sequence data are available are PKC-B (types I and II), PKC-~ (par ial s~quence) and P~ . Antisense oligonucleotides which are likely to be e~fective inhibitor~ of PKC are identified below.
These oligonucleotides are synthesized as in Example l, and can ba screened as in Examples 2 and 3, using appropriate antibodies where available. Alternatively, a reporter gene , assay system can be established, transiently co-expressing the 15 desired isozyme of PKC with luciferase under the influence of the TPA--responsive enhancer or other suitable promoter. PKC
expres~ion is then assayed by measuring luci~era5e activity u~ing standard procedures: luciferase is extracted from cell5 by lysis with the detergent Triton X-l00, as described by 20 Greenberg, MoE~ I in urrent Protocols in Molecular Bioloq~, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J~G. Seidman and K. Strahl, Eds., John Wiley and Sons, NY
(1~87). A Dynatech ~Ll000 luminometer is used to measure peak luminescence upon addition of luciferin ~Sigma) to 625 ~M.
25 P~C~B, typ~s I a~
Sequence data are from Kubo et al., FEBS Lett. l987, 223 138-142; ~sequences~ are numbered from the first 5' ba~e sequenced on the cDNA. PKC-B types I and II are the result of , ~ al ernative mRNAisplicing of ~a single gene`~productl Th`is 30 result~ in prot~ins with identica~ amino termini (5' end of the mRNA); however,~there;is sequence diverg~nce in the carboxy termini (3f end of the mRNA). The following oligonucleotides, targeted to t~e translation initiation codon, are expected to : modulate expre~sion of both PKC-B types I and II:
:: :
:
: ~ : :
WO~3/19203 PCT/US93/0~213 , .
~3 - 18 - f``
OLIGONUCLEOTIDES TARGETED TO PKC-B TYPES I AND II I .:
SE~ I~ SEQUENC~ ~ARGET
21 CAT C~T GCG CGC GGG ~AG ~C Translation init. codon 113 94 ;
10 24 CTC TCC TCG CCC TCG ~TC GG " " .:
~83 164 .
The following antisense oligonucleotides are targeted to the 3'-untranslated region of PKC-B type I only~
OLIGONUCLEOTIDES TAR~ETED TO PKC-B TYPE I
SEQ ID SEQUENCE TARGET
TGG AGT TTG CAT TCA CCT AC 3' Untranslated 26 AAA GGC CTC TAA GAC ~AG CT " ~Y
2285 2266 ..
27 GCC AGC ATG TGC ACC GTG AA " " :.
2250 2231 .
28 ACA CCC CAG GCT CAA CGA TG " " ::~
~186 2167 2~ CCG AAG CTT ACT CAC AAT TT
2569 2550 .
; ~The foliowing antisense oligonucleotides are targeted to the 3' untranslated ~egion of PKC-B Type II only~
' ,.
W093~19203 ` 2 ~ 3 2 0 9 ~1 PCT/US93/02213 -- lg -- , OLIGONUCLEOTIDES T ~GETED TO PKC-B TYPE II
~Q_L~ SEQUE~~ TARGET
ACT TAG CTC TTG ACT TCG GG 3' Untranslated ~' 31 ATG CTG CGG AAA P~TA AAT TG " " :-` 2420 2401 32 ATT TTA TTT TGA GCA TGT TC " "
26~3 ~2644 ~ ~
,.
2$43 ~ 2824 ! .
:.
,' "~, 34 CCC ATT CCC ACA GGC CTG AG~ "
~ 3137 3118 ,~:
PRC-~:
15 ~ ~ ~~nquence data are from Cou~ ~ et al., Science 1986, ~.; 233,~8 ~66;~sequences are num~ from ~he first 5~: base equen~ ~ in~the~cDNA. The full s~ ~nce is not available: the extreme~ 3' end of the open r~ading frame~ and the 3' unt~ansl~ated~r~gi~on~are~missing.~ Con~equent1y these reglons~
20 ~are~not presently~available~as ant~isense targets.
TAB~E 5 OLI~60NU OEEOTIDES TARGETED TO~PKC~
S~EO_~ID~ EQ~S~ TARGET
` 35~ C G AGC~GCG~;CCA~GGC ~GG~GA ;5~'~Untranslated~
36 CCT TTT CCC ~GA CC~ GCC AT Translation init. codon : : : ` ~ : : ~ : ; ',:
37 ; GGC~C~C AGA~AAC~;GTA G~GG 5' o~ sta~t cod~n 0~38~ GGA~T~C;~TGO~CTT~ TCT TGG~ GG ~`~ 5'~Untranslated 39 ~ AG`~CA TGG~CCC ~AG~AAA~CG Translation init. ~don WO 93/1~203 PCI'/US93/02213 ~,~3~09~ - 20 1~
- P~
Sequence data for PKC-~7 are from Bacher and colle~gues [Bacher et al., Mol. Cell. Biol. 1991, 1~, 126-133]. They assign their i50zyme the name PKC-~; however the sequence is 5 almc~st identical to that. of mouse PKC-?1, so t~e latter nomenclature is used here f or consistency O Sequences are numbered from the f irst S ' base sequenc:ed in the cDNA.
OLIGONUCLEOTIDES TARGETED TO PKC~
10 ~2 SEQUENCE TARGET
CGA CAT GCC GGC GCC GCT GC Translation init. codon 172 lS3 41 CAG ACG ~CA TGC CGG CGC CG
lS 42 GCC TGC TTC GCA GCG GGA GA "
86 ' 67 Ds 5 ~CT CAC CGA TGC GGA CCC TC 1- .. , 4 6 ~TT GP~A CTT CAT GÇ;T GCC AG " 1l :
193 ~74 25 47 TCT CAC TCC CCA TAA GGC TA 3 ' Untranslated 204~ 2027 4 8 TTC CTT TGG t;TT CTC GT(; CC
4 9 TT~ CAT CCT TCG ACA GAG TT " " J
. ~
S O AGG ~TG ATG CTG G&A AÇ;G TC " '~
2300 2~81 51 GTT CTA AGG CTG ATG CTG GÇ;
~306 :~87 . . .
Ex~pl~ S Oligo~u~leotid~ i~hibition of PXC i8 aO~e ~epe~d~t Four oligo~ucleotides shown in Figure 1 to be active against PKC-~ were characterized fur~her. ~ose response studies of ISIS 3520 (SEQ ID NO:l), 3521 (SE~ ID N0:2), 3522 (SEQ ID N0:3) and 3527 (SE~ ID N0:5) using the immunoblotting assay describ~d in Example 3 demonstrated that all had dose-depend~nt activity against PKC-~ protein expression. ISIS
3521, 3522 and 3527 had IC50 values of 100-200 ~M, and all maximally inhibited PKC expression at 500 nM. This is shown in Figure 2~ ISIS 49~5 (SEQ ID N0: 52), a scrambled control oligonucleotide having the same base composition as ISIS 3527, was without effect.
xampl~ 6 8y~th~si~ of 2~-O-~ethyl pho~Phorothioate oligo~u~leotides 2~0-methyl phosphorothioate oligonucleotides were synthesized using 2'-0-methyl B-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham MA) and the standard cycle for unmodified oligonucleotides, except the wait step a~ter pulse delivery of ~tetrazole and base was increased to 360 seconds. ~ The 3'-base used:to start the synthesis was a 2'-~deoxyribonucIeotide.
~P1R 7 B~foct of 2'-0-~ethyl~ted ver~io~ of I8IB 3522 on C-~ prot~ thQsi~ ~
~ A uniform1y; ~2'-0-methyl modified phosphorothioate oligonucleotide, ISIS 4649, having the:same sequence as the ~phosphorothio~te oligonucleotide ISIS 3522 (SEQ ID N0:3), was able~:to inhibit~PKC protein ~synthesis (demonstrated by immunoblotting as described in ~xample 3),Iwith an~ICsO~o~ less than 300 nM. This is shown in Figure:3.
~mpl-~8~ f;~ect~of oligoDuc1~o~id~s o~ PRC-~ s~NA expre~sio~
~ To~determi~e~ the:~effe:cts~of ol~igonucleotides on PKC-~mRNA :levels,~ A54~cel~ls~were~reated with oligonucleotides a~
~the~ ;indicated~ concentr~ation in the~ presen~e oP cationic ;liposomes for~four hours.~ Tota1~cellular RNA was isolated from
: 193 : 174 g ~CA GAG GCT GGG GAC ATT GA Internal (Cl) domain . -~
: 480 461 25~:10 GGG CTG GGG ~GG~TGT TTG TT 3' Untranslated 2080 ~ ~ 2061 ~ :
~` ` ll . CAC TGC GGG G~G GGC TG~ GG 3t Untranslated :: 20~ 20~9 : ~:
l2 ~ AGC CGT GGC CTT ~ ATT TT j 3' Untranslated 301 i 2137 ~ 2118 :
~:~:: 13~ ~TT TTC AG~ CCT~CCA TAT ~G 3' Untranslated 2168~ 2149 l4 AAG AGA GAG A~C:CTG~AAC AG 3' Untranslated .
:: 2217: ~ 2198 35 15 ~GAT AAT GTT CTT G&~ TGT ~A 3' Untranslated 2235 ~ :~2216 ;
WV93/19~03 Pcr/uss3/o22l3 '1~3~ 16 ~
16 ATG GGG TGC ACA AAC TGG GG Internal (C3) domain 17 GTC AGC CAT GGT CCC CCC CC Translation init. codon 18 CGC CGT GG~ GTC GTT GCC CG Internal ~Vl) domain 63 44 -`
i9 TCA AAT GGA GGC TGC CCG GC Internal (C3) domain .
TGG AAT C~G ACA CAA GCC GT 3' Untranslated 21~1 2132 .
E~a~ple 3 I~u~oblot ~ay for P~C ~xpr~3~ion - Cell extracts were electrophoresed on 10% SDS-PAGE mini-gels. The resolved proteins were transferred to I~mobilon-P
membrane (Millipore, Bedford MA) by electrophoretic transfer }5 and the membrane was blocked for 60 minutes in TBS (Tris-HCl pH
7.4, 15Q mM NaC1) contain~;.ng 5% nonfat milk. The membrane was then incubated for 16 hours at 4C with monoclonal antibodies raised against PKC-~ (UBX, Lake Placid NY) diluted to 0.2 ~gjml in TBS containing 0.2$ nonfat milk. This was followed by three 20 washes in TBS plus 0.2% nonfat milk. The membrane was ~hen incubated ~or one hour with l2sI-labelled goat anti-mouse secondary antibody (ICN Radiochemicals, Irvine CA). Membranes were then washed extensively in TBS plus 0.2% nonfat milk.
: Bands were ~isualized and quantit~ted using a Phosphorimager (:Molecular Dynamics, Sunnyvale, CA). PKC-~ appears as a single band with a molecular weight of 80 kD.
Each oligonucleotide was tested three times, in trîplicate, and the results of the experiments were normalized a~ainst percentage of protein present as compared to cells ~0 which were not~treated with oligonucleotide (Figure 1). The j~.
:~~ive most e~fective oligonucleotides target the AUG start codon ~:
and regions slightly upstream and downstream from it ~oligos 1, --3;, 17, 7, 6)o The next most effective oligonucleotides are targeted toward the 3' unt~anslated region of the RN~ ~oligos 2, 5j 14).
.,:
:
W093/~9203 2 ~ ~ 2 3 9 ~ PCT/US93/02213 ~ampl~ ~ Ot~r i~o~y~3 of P~C
It was found that the most ef~ective sequences for antisense targeting of h~man PKC~ are those surrounding the transl~tion initiation codon and the 3' untranslated region.
It is believed that these sequences would also be effective targets for oligonucleotides directed against other isozymes of PKC. The other isozymes of human PKC for which sequence data are available are PKC-B (types I and II), PKC-~ (par ial s~quence) and P~ . Antisense oligonucleotides which are likely to be e~fective inhibitor~ of PKC are identified below.
These oligonucleotides are synthesized as in Example l, and can ba screened as in Examples 2 and 3, using appropriate antibodies where available. Alternatively, a reporter gene , assay system can be established, transiently co-expressing the 15 desired isozyme of PKC with luciferase under the influence of the TPA--responsive enhancer or other suitable promoter. PKC
expres~ion is then assayed by measuring luci~era5e activity u~ing standard procedures: luciferase is extracted from cell5 by lysis with the detergent Triton X-l00, as described by 20 Greenberg, MoE~ I in urrent Protocols in Molecular Bioloq~, F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.A. Smith, J~G. Seidman and K. Strahl, Eds., John Wiley and Sons, NY
(1~87). A Dynatech ~Ll000 luminometer is used to measure peak luminescence upon addition of luciferin ~Sigma) to 625 ~M.
25 P~C~B, typ~s I a~
Sequence data are from Kubo et al., FEBS Lett. l987, 223 138-142; ~sequences~ are numbered from the first 5' ba~e sequenced on the cDNA. PKC-B types I and II are the result of , ~ al ernative mRNAisplicing of ~a single gene`~productl Th`is 30 result~ in prot~ins with identica~ amino termini (5' end of the mRNA); however,~there;is sequence diverg~nce in the carboxy termini (3f end of the mRNA). The following oligonucleotides, targeted to t~e translation initiation codon, are expected to : modulate expre~sion of both PKC-B types I and II:
:: :
:
: ~ : :
WO~3/19203 PCT/US93/0~213 , .
~3 - 18 - f``
OLIGONUCLEOTIDES TARGETED TO PKC-B TYPES I AND II I .:
SE~ I~ SEQUENC~ ~ARGET
21 CAT C~T GCG CGC GGG ~AG ~C Translation init. codon 113 94 ;
10 24 CTC TCC TCG CCC TCG ~TC GG " " .:
~83 164 .
The following antisense oligonucleotides are targeted to the 3'-untranslated region of PKC-B type I only~
OLIGONUCLEOTIDES TAR~ETED TO PKC-B TYPE I
SEQ ID SEQUENCE TARGET
TGG AGT TTG CAT TCA CCT AC 3' Untranslated 26 AAA GGC CTC TAA GAC ~AG CT " ~Y
2285 2266 ..
27 GCC AGC ATG TGC ACC GTG AA " " :.
2250 2231 .
28 ACA CCC CAG GCT CAA CGA TG " " ::~
~186 2167 2~ CCG AAG CTT ACT CAC AAT TT
2569 2550 .
; ~The foliowing antisense oligonucleotides are targeted to the 3' untranslated ~egion of PKC-B Type II only~
' ,.
W093~19203 ` 2 ~ 3 2 0 9 ~1 PCT/US93/02213 -- lg -- , OLIGONUCLEOTIDES T ~GETED TO PKC-B TYPE II
~Q_L~ SEQUE~~ TARGET
ACT TAG CTC TTG ACT TCG GG 3' Untranslated ~' 31 ATG CTG CGG AAA P~TA AAT TG " " :-` 2420 2401 32 ATT TTA TTT TGA GCA TGT TC " "
26~3 ~2644 ~ ~
,.
2$43 ~ 2824 ! .
:.
,' "~, 34 CCC ATT CCC ACA GGC CTG AG~ "
~ 3137 3118 ,~:
PRC-~:
15 ~ ~ ~~nquence data are from Cou~ ~ et al., Science 1986, ~.; 233,~8 ~66;~sequences are num~ from ~he first 5~: base equen~ ~ in~the~cDNA. The full s~ ~nce is not available: the extreme~ 3' end of the open r~ading frame~ and the 3' unt~ansl~ated~r~gi~on~are~missing.~ Con~equent1y these reglons~
20 ~are~not presently~available~as ant~isense targets.
TAB~E 5 OLI~60NU OEEOTIDES TARGETED TO~PKC~
S~EO_~ID~ EQ~S~ TARGET
` 35~ C G AGC~GCG~;CCA~GGC ~GG~GA ;5~'~Untranslated~
36 CCT TTT CCC ~GA CC~ GCC AT Translation init. codon : : : ` ~ : : ~ : ; ',:
37 ; GGC~C~C AGA~AAC~;GTA G~GG 5' o~ sta~t cod~n 0~38~ GGA~T~C;~TGO~CTT~ TCT TGG~ GG ~`~ 5'~Untranslated 39 ~ AG`~CA TGG~CCC ~AG~AAA~CG Translation init. ~don WO 93/1~203 PCI'/US93/02213 ~,~3~09~ - 20 1~
- P~
Sequence data for PKC-~7 are from Bacher and colle~gues [Bacher et al., Mol. Cell. Biol. 1991, 1~, 126-133]. They assign their i50zyme the name PKC-~; however the sequence is 5 almc~st identical to that. of mouse PKC-?1, so t~e latter nomenclature is used here f or consistency O Sequences are numbered from the f irst S ' base sequenc:ed in the cDNA.
OLIGONUCLEOTIDES TARGETED TO PKC~
10 ~2 SEQUENCE TARGET
CGA CAT GCC GGC GCC GCT GC Translation init. codon 172 lS3 41 CAG ACG ~CA TGC CGG CGC CG
lS 42 GCC TGC TTC GCA GCG GGA GA "
86 ' 67 Ds 5 ~CT CAC CGA TGC GGA CCC TC 1- .. , 4 6 ~TT GP~A CTT CAT GÇ;T GCC AG " 1l :
193 ~74 25 47 TCT CAC TCC CCA TAA GGC TA 3 ' Untranslated 204~ 2027 4 8 TTC CTT TGG t;TT CTC GT(; CC
4 9 TT~ CAT CCT TCG ACA GAG TT " " J
. ~
S O AGG ~TG ATG CTG G&A AÇ;G TC " '~
2300 2~81 51 GTT CTA AGG CTG ATG CTG GÇ;
~306 :~87 . . .
Ex~pl~ S Oligo~u~leotid~ i~hibition of PXC i8 aO~e ~epe~d~t Four oligo~ucleotides shown in Figure 1 to be active against PKC-~ were characterized fur~her. ~ose response studies of ISIS 3520 (SEQ ID NO:l), 3521 (SE~ ID N0:2), 3522 (SEQ ID N0:3) and 3527 (SE~ ID N0:5) using the immunoblotting assay describ~d in Example 3 demonstrated that all had dose-depend~nt activity against PKC-~ protein expression. ISIS
3521, 3522 and 3527 had IC50 values of 100-200 ~M, and all maximally inhibited PKC expression at 500 nM. This is shown in Figure 2~ ISIS 49~5 (SEQ ID N0: 52), a scrambled control oligonucleotide having the same base composition as ISIS 3527, was without effect.
xampl~ 6 8y~th~si~ of 2~-O-~ethyl pho~Phorothioate oligo~u~leotides 2~0-methyl phosphorothioate oligonucleotides were synthesized using 2'-0-methyl B-cyanoethyldiisopropyl-phosphoramidites (Chemgenes, Needham MA) and the standard cycle for unmodified oligonucleotides, except the wait step a~ter pulse delivery of ~tetrazole and base was increased to 360 seconds. ~ The 3'-base used:to start the synthesis was a 2'-~deoxyribonucIeotide.
~P1R 7 B~foct of 2'-0-~ethyl~ted ver~io~ of I8IB 3522 on C-~ prot~ thQsi~ ~
~ A uniform1y; ~2'-0-methyl modified phosphorothioate oligonucleotide, ISIS 4649, having the:same sequence as the ~phosphorothio~te oligonucleotide ISIS 3522 (SEQ ID N0:3), was able~:to inhibit~PKC protein ~synthesis (demonstrated by immunoblotting as described in ~xample 3),Iwith an~ICsO~o~ less than 300 nM. This is shown in Figure:3.
~mpl-~8~ f;~ect~of oligoDuc1~o~id~s o~ PRC-~ s~NA expre~sio~
~ To~determi~e~ the:~effe:cts~of ol~igonucleotides on PKC-~mRNA :levels,~ A54~cel~ls~were~reated with oligonucleotides a~
~the~ ;indicated~ concentr~ation in the~ presen~e oP cationic ;liposomes for~four hours.~ Tota1~cellular RNA was isolated from
9~03 P~T/US93/02213 2~32 9 ~ - 22 - ~
cells by lysis in 4M yuanidinium isothiocyanate followed by a cesium chloride gradient. Total RNA (15-30 ~g) was resolved on l.2% agarose gels contalning l.~% formaldehyde and transferred to nylon membranes. The mQmbranes were then hybridized with S bovine PXC-~ cDNA obtain~d from the ~TCC (Bethesda, MD). The cDNA was 32p radiolabelled with [~_32p] dCTP by random primer labeling using a commarcially available kit (Promega3 according to the manufacturer's instructions. The filters were hybridized for 60 minutes in Quikhyb solution ~Stratagene, San l0 Diego, CA) at 68C. This was followed by two low stringency washes (2x SSC/0.1% SDS) at room temperature and two high stringency washes (0.lx SSC/0.1% SDS at 60C. Hybridizing bands were visualized and quantitated using a phosphorimager.
The blots were then stripped of radioactivity by boiling and l5 reprobed with a 32P-la~eled glycerol-3-phosphate dehydrogenase (G3PDH) probe ~Clontech) to confirm equal RN~ loading.
Northern analysis of total RNA from A549 cells using the PKC-~-specific cDNA probe revealed two major hybridizing transcripts approximately 8.5 kb and 4.0 k~ in size. Typically 20 these are exp~essed in a two-to-one ratio with the larger transcript predominating. When A549 cells were treated with antisense oligonucleotides at ~00 nM for four hours in the presence of DOTMA and then incubated for an additional 20 hours, level~ of both transcripts were decreased. This is shown in Figure 4. The greatest decrease was seen with oligonucleotides ISIS: 3521 (SEQ ID NO:2) and 3527 (SEQ ID
NO:5), bsth specifically hybridizablP with 3'-untranslated sequences. These reduced amounts of PKC-~ mRNA by g0-95%.
ISIS 3522 (SEQ ID NO:3), targeted to the translation start 39 cod~n, decreased PKC-~ mRNA levels by 80%, and ISIS 35l20 (S Q
ID NO: l) decreased PKC mRNA by 40%. All oligonucleotides had ICso values of approximately 200 nM for PKC mRNA reduction.
Scrambled contral oligonucleotide showed no effect at this concentration.
The kinetics of antisense oligonucleotide decrease in P~C
mRN~ levels were similar for the four oligonucleotides tested.
Within four hours of oligonucleotide addition, 60-70~ of W093/~9~03 2 1 3 2 0 ~ ~ P~T/US93/02213 .. .
maximal PKC RNA reduction occurred, and maximal reduction occurred 12-24 hours after oligonuclaotide addition.
B$~p~o 9 ~pe~ifi~ bitio~ of ~C- by a~tise~
oliqo~ucl~oti~
A549 cells were ~hown to normally express PKC-~ and -~ in addition to PKC-~. The sp~ciicity of inhibition of the PKC-~ isozyme by antisense oligonucleotides targeted to this isozyme was demonstrated in these cells by immunoblotting after treatment wikh ISIS 3521 (SEQ ID NO:2, ISIS 3522 (SEQ ID NO:3)
cells by lysis in 4M yuanidinium isothiocyanate followed by a cesium chloride gradient. Total RNA (15-30 ~g) was resolved on l.2% agarose gels contalning l.~% formaldehyde and transferred to nylon membranes. The mQmbranes were then hybridized with S bovine PXC-~ cDNA obtain~d from the ~TCC (Bethesda, MD). The cDNA was 32p radiolabelled with [~_32p] dCTP by random primer labeling using a commarcially available kit (Promega3 according to the manufacturer's instructions. The filters were hybridized for 60 minutes in Quikhyb solution ~Stratagene, San l0 Diego, CA) at 68C. This was followed by two low stringency washes (2x SSC/0.1% SDS) at room temperature and two high stringency washes (0.lx SSC/0.1% SDS at 60C. Hybridizing bands were visualized and quantitated using a phosphorimager.
The blots were then stripped of radioactivity by boiling and l5 reprobed with a 32P-la~eled glycerol-3-phosphate dehydrogenase (G3PDH) probe ~Clontech) to confirm equal RN~ loading.
Northern analysis of total RNA from A549 cells using the PKC-~-specific cDNA probe revealed two major hybridizing transcripts approximately 8.5 kb and 4.0 k~ in size. Typically 20 these are exp~essed in a two-to-one ratio with the larger transcript predominating. When A549 cells were treated with antisense oligonucleotides at ~00 nM for four hours in the presence of DOTMA and then incubated for an additional 20 hours, level~ of both transcripts were decreased. This is shown in Figure 4. The greatest decrease was seen with oligonucleotides ISIS: 3521 (SEQ ID NO:2) and 3527 (SEQ ID
NO:5), bsth specifically hybridizablP with 3'-untranslated sequences. These reduced amounts of PKC-~ mRNA by g0-95%.
ISIS 3522 (SEQ ID NO:3), targeted to the translation start 39 cod~n, decreased PKC-~ mRNA levels by 80%, and ISIS 35l20 (S Q
ID NO: l) decreased PKC mRNA by 40%. All oligonucleotides had ICso values of approximately 200 nM for PKC mRNA reduction.
Scrambled contral oligonucleotide showed no effect at this concentration.
The kinetics of antisense oligonucleotide decrease in P~C
mRN~ levels were similar for the four oligonucleotides tested.
Within four hours of oligonucleotide addition, 60-70~ of W093/~9~03 2 1 3 2 0 ~ ~ P~T/US93/02213 .. .
maximal PKC RNA reduction occurred, and maximal reduction occurred 12-24 hours after oligonuclaotide addition.
B$~p~o 9 ~pe~ifi~ bitio~ of ~C- by a~tise~
oliqo~ucl~oti~
A549 cells were ~hown to normally express PKC-~ and -~ in addition to PKC-~. The sp~ciicity of inhibition of the PKC-~ isozyme by antisense oligonucleotides targeted to this isozyme was demonstrated in these cells by immunoblotting after treatment wikh ISIS 3521 (SEQ ID NO:2, ISIS 3522 (SEQ ID NO:3)
10 or their scrambled control oligonucleotides, ISIS 4559 (SEQ ~D
NO: 53) and 4608 (SEQ ID NO: 54~. A549 cells were treat~d for four hours with oligonucleotide (either 400 nM or 300 nM) and ,. .
DOTMA, washed, and allowed to recover for 48 hours. Cells were then treated again with oligonucleotide (250 nM) and DOTMA, ~-15 washed and allowed to recover for a further 20 hours. Cell `-extracts were electrophoresed, transferred to membrane and .~-blocked as described in Example 3. ~he membrane was then , .
treatsd overni~ht with either of the following antibodies diluted in 0.5% nonfat milk in TBS: anti-PKC-~, B or 20 :monoclonal (Upstate Biochemicals, Inc.3, 1 ~g/ml; anti PKC-~ or ..
-~ polyclonal (Gibco BRL~ 2000 dilution; anti-PXC-~ [Huwiler et al., Bioche~. J. 199g, 279, 441~445], 1:4000 dilution; or L ~=
anti-PKC~ 4000 dilution. Anti~ody incubation was followed .~.
by three washes in TBS containing 0.1~ nonfat milk and the 25 memb~ane was then incubated with 5 ~Ci 1~sI-goat anti~rabbit or anti-mouse antibody (ICN Radiochemicals, Irvine CA) for one :
hour. Membranes were washed extensively and the labelled .--~
proteins were visualized and quantitated using a phosphorimager (MO1QCU1ar Dynamics, CAj~
'` 30 Analysis of the PKC isozymes expressed after -: ~ligonucleotide ~reatment revealed that le~els of PKC~ and ~:
-~ were un hanged by treatment with either of the antisense oligonucleotides or their scrambled controls. The antisense !~^
oligonucleotides decreased PKC-~ expression by 70-r80%, while scrambled controls had no effect.
,.
1, .
W093/~9203 PCT/US93/02213 ~ampl~ 10 Ef~ect of oligonualeoti~s3 on A549 cell proli~ratio~
A549 cells were plated in 6-well plates at a concentration of 4000 cells per well~ After 24 hours cells 5 were washed three times in.DMEM and then oligonucleotides were added to t~e required concentration in the presence of 20 ~g/ml DOTMA for four hours. Cel:ls were then washed once in DMEM plus 5~ FCS and allowed to grow in DME~ plus 5% FCS for a further 72 hours. At this time cells were washed twice in PBS, removed 0 from the dishes with trypsin and counted with a hemocytometer.
Inhibition of A549 cell proliferation by ISIS 3S21 and 3527:was seen after a single oligonucleotide dose of 250-500 nM. This is æhown in Figure 5.
~xample ll I~hibition of PRC~ by chim~ric ~nti~ns~
oligonu¢leoti~es Chimeric phosphorothioate oligonucleotides having "deoxy : gapæ:" of 4 to 8~deoxynucleotide~ in an otherwise 2'-0-methyl oli~onucleotide were tested for ability to decrease PKC~ m~NA
levels as:des~ribed in Example 8. Chimeric oligonucleotides 20 ~were identical in sequence to ISIS 3521 (SE~ ID N0:2) ISIS 3522 (SEQ ~ID N0: 3) and~ISIS 3527 lSEQ ID N0:5). Chimeric ` oligonucleotides are:shown in Table 7:
:
: :
WO 93/19203 2 1 3 ~ O ~ ~ PC~ /us93/o22l3 ~
PKcA GAPPED OLI&ONUCLEOTIDES
Boldl indicates 2 ' -O-methyl nucleotides All oligonuc:leotide are phosphorothioat2s OLIGO SEQUl :NCIS DESCRIPTION SEQ ID NO
3521 GTTCTCGCTGGTGAGTTTCA Full P=S
5357 ~ C$C:GCTGGTGAGTTTCA Full P=S 8-deoxy gap 2 S361 G~T~CTC~3CTGGTGAGT~TC~ Full P--S 6-deoxy gap 2 53~0 tS~CTC~3CTGGTGA~rTTC~ Full P=S 4-deoxy gap 2 10 3522 A~ACGTCAt:CCATGGTCCC Full P=S 3 535~ AAAACGTC~GCCA~GGq!CCC Full P=S 8-deoxy gap 3 5350 ~CG~CAGCCA~GTCCC Full P=S 6-deoxy gap 3 S351 AAa;~CG~rCA~;CCP~GG~t~CC Full P-S 4-deoxy gap 3 3527 GAG~CCCTGAACAGTTGATC Full P=S 5 15 5240 ~;~GACCCTGAAC~ :;TTG~TC Full P=S 8~deoxy gap 5 5208 GAG;~CS~C~GAAC.~TTGATC Full P=S 6-deoxy gap 5 5038 GAG~CCCTGAACAt~TTGATC Full P=S 4-deoxy gap 5 The 8-deoxy ga~pped oligonucleotides were able to lower PKC~ ~ NA le~rels by at least 85%. Activity of 8-deoxy gapped 20 oligonucleotides compared to ungapped oligonucleotides having the same sequence is shown in Figure 6A, 6B and 6C. Two of the 6-deoxy ~apped oligonucleotides ( ISXS 53 61, SEQ ID NO: 2 . and ISIS 5350, SEQ ID NO:3 ) were able to lower PKCc~ mRNA by greater than 50%, and one of the 4-~eoxy gapped 25 oligonucleotides (I5IS 3522, SEQ I1:) NO: 3) was able to inhibit PKCa~ mRNA by approximately 85%. Activity vs. d.eoxy gap length f or oligos having SEQ ID NC): 2, SEQ ID NO: 3 ~nd SEQ ID ~O: 5 is shown in Figure 7.
I , i , , I . ~ j .
~a~pl~ 12 Oligonucleot~e tr~t~t of hu~a~ tu~or eell~
~u~a ~ic~ 'f Human Iung carcinoma A549 cells were harves~ed and 5 x : 106 cells were inje~ted subcutaneously into the inside hind leg of nude mice. Palpable tumors davelop in approximately one month~ Antisense oligonucleotides ISIS 3521 and 3527 are adminis~ered to mice intraperitoneally at two doses, 1 and 10 :
::
mg/kg body weight, every other day for six wee3cs. Mice are monitored for tumor gro~rth and after six weeks the t~mors are excised and the expression of PKCc~ :~s de~ermined ~y Northern blot and immunoblot.
Although preferred embodiments of the invention have been described herein~
it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
-~ : :
.
: ~ ' : .
-SEQUENCE LISTING
(1) GENERAL INFORMATION:
(.i) APPLICANT: Nicholas Dean, C. Frank Bennett (ii) TITLE OF INVENTION: Oligonucleotide Modulation of Protein Kinase C
(iii) NUMBER OF SEQUENCES: 54 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz & Norris (B) STREET: One Liberty Place - 46th Floor ~ (C) CITY: Philadelphia (D) STATE: PA
(E~ COUNTRY: USA
(F) ZIP: 19103 (v) COMPUTER~READABLE FORM:
(A~ MEDI~M TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE
(B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
~D) SOFTWARE: WORDPERFECT 5.0 ;~ (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a (B) FILING DATE: herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A3 APPLICATION NUMBER: 852,852 (B) FILING:DATE: March 16, 1992 ~: ~viii)~ATToRNEY/AGENT INFORMATION:
; ~ ~ (A):N~ME: Jane Massey Licata (B) REGISTRATION NUMBER: 32,257 ,.,r~
;`::: :: `:
: : :
W093/19~03 PCT/U~93/02~13 ~3~09~ - 28 -(C) REFERENOE/DOCKET NUMBER: ISIS-0872 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568~3l00 (B) TELEFAX: (2l5) 568-3439 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS~ single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
CCCCA~CCAC CTCTTGCTCC 20 (2) INFORMATION FOR SEQ ID NO: 2:
~i) SEQUENCE CHARACTE~ISTICS:
~: (A) LENGTH:~20 ~B) TYPE: nucleic acid (C) STRANDEDNESS: single : ~ (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes ~(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2~ INFORMATION FOR ~EQ;ID NO: 3:
~k ~ ~ I
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH:~:20 (B) TYPE: nucleic acid : ~ :
~ (C)~ STRANDEDNESS:~single ::: : : : ` :
~ (D) TOPOLOGY::linear ~ ~ .
WO 93/19203 PCr/US93/OZ213 ( iv) ANTIWSENSEq yes ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 3:
~ GTCAG CCATGGTCCC 2 0 ( 2 ) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACrERISTï::S:
( A ) LENGTH: 2 0 (B) TYPE: nuclei :: acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( iv) ANTI~SENSE: yes (xi) SEQUE~CE DESCRIPTION: SEQ Il) NO: 4:
GGATTCACTT CCACTGCGGÇ: 2 0 t 2 ) INFORM~TION FOR SEQ ID NO: 5 ~ ;
( i ) SEQUENCE C~ACTERISTICS:
( ~ ) LENGTEI: 2 O
(B) TYPE: nucleic acid ~ ) STRA~DEDNESS: single ( D`) TOPOLOGY: l inear t i~) ANTI-SENSE: yes ( xi ) S~QUENCE: DESC~IPTION: SEQ ID NO: 5:
GACCCTGA P.CP~GTT :;ATC 2 û
.
( 2 ) XNFORMATIOM FQR ^SEQ TD NO: 6:
(i) 5EQUEN~E C~ARACTERISTICS:
1~ ' .
tA) LENGTH: 20 s (B) TYPE. nucleic ac:id (C) ST~DEDNESS: single ( D ~ TOPOLOGY: l inear ( iv~) ANTI~5ENSE: yes W093~19203 PCT/US93tO2213 ~ 0 9 ~ 30 _ ' (xi) SEQUEMCE DESCRIPTION: SEQ ID NO: 6:
CCCG~GAA~A CGT~AGCCAT 20 (2) INFO~MATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) ST ~ DEDNESS: single (D3 TOPOLOGY: linear (iv) ~NTI-SENSE: yes : (xi) SEQUENCE DESCRIP~ION: SEQ ID NO: 7:
(2) INFORMATION FOR SEQ ID NO: 8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B~ TYPE: nucleic acid (C~ STR~NDEDNESS: single : ~ . (D) TOPOLOGY: linear (iv3 ~NTI-SENSE: yes : ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR:SEQ ID NO: 9:
SEQUENCE:C~ARACTERISTICS:
(A)~LENGTH~:~20 ~B)~;~TYPE: nucleic acid (C~) STRANDEDNESS~: single (D)~TOPOLOGY: linear (iv): ~NTI-SENS ^ yes - ; (xi~) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
.
, ~
W093/19203 2 ~ 3 ~ ~ 9 ~ PCT/US93/02213 1 ' - 3~ ~ :
GCAG~GGCTG GGGACATTGA 20 (2) INFORMATION FOR SEQ ID NO l0: ~ -(i) SEQUENCE CH~RACTERISTICS:
~A) LEN&TH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
t2) INFORMATION FOR SEQ ID NO: ll: ` ~.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 ; ~B~ TYPE: nucleic acid (C3 ST~ANDEDNESS: single (D) TOPOLOGY: linear ~iv) ANTI~SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:
: CACTGCGGGG AGGGCTGGGG 20 (2) INFQRMATION FOR SEQ ID NO: 12: i (i) SEQUENCE CHARACTERISTICS: :
(~) LENGTH: 20 .
tB) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOhOGY~: linear ~iv) ANTI-SENSE- yes .
:~ ~ (xi~ SEQUENCE~DESCRIPTIVN: SEQ ID NO: 12:
WO93/l9203 PCT/US93/02213 :
~o94 - 32 (~) ORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv3 ANTI SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
-~2) INFORMATION FOR~SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 20 (B) TYPE: nucleic acid:
(C) 5TRANDEDNESS: single , : (D) TOPOLOGY: linear ~iv) ~NTI-SEN~E: yes (xi) SEQUEN~E DESCRIPTION: SEQ ID NO 14: . ~' A~GAGAGAGA CCCTGAACAG 20 :
(2)~ INFORMATION~FOR SEQ ID NO: lS~
SEQUENCE CHARACTERISTICS~
(A) ~ENGT~: 20~
B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ q (D)~TOPOLOGY~ linear~
(iv)~ANTI-SENSE:~yes~
SEQUEN~E~E~RIPTION~ SEQ ID~:NO::15 GATAATGTTC~TT6GTTGTAA 20 W093/19203 2~32~ PCI/US93/02~13 (2) INFORMATION FOR SEQ ID NC): 16:
i ) SEQtJENC13 C~CTERISTICS
(A~ L:~SNGTH: 20 (B~ q~YPE: nucleic acid t ;~
( C) STRANDED~ESS: single ` .
~ D ) TOP~;)LO :;Y: 1 inear - . .
( iv~ ANTI SENSE: yes ~xi) SEQUENCE r)ESCRIPTION: SE~ ID NO: 16:
ATG~:;GGTGCA CA~CTGGGG 2 0 . ~;
. . , .2 ) INFORMATION FOR EQ ID NO : 17:
i ) SEQUENCE CHARACTERISTICS:
(A) ~ENt;TH: 20 (B) TYPE: nucleic acid . ~ .
(C) ST~DEDNESS: single ~-(D) ~OPOLOGY:~ linear .
~ iv) ANT~-SENSE: yes . . ; ;~
(xi) SEQUENCE DESCRIPTIC1N: S~Q ïD NO: 17:
~TCAGCCATG GTCCCCCCCC 2 0 : .
( 2 ) INFORM~TION FOR SEQ ID NO: 18:
( i) SEQIJ:E:NCE CHARACTERISTICS:
( A ~ LENGTH : 2 0 ~;
~.
(B) TYP~: nuclei :: acid i ( C) STRANDEDNESS: siYIgle (D) TOPOLOGY: linear ~, ( iv~ ANTI~SENSE: y~s : ~
: 1 : ~ ( Xi ) SEQUENC:E DESCRIPTI~ON: SEQ ID NC): 18: ~
:
~ 3 ~ ~ 3~ ! :
(2) INFORMAT~ON FOR SEQ ID NO: l9:
~i~ SEQUENCE C~ARACTERISTICS~
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv) ~NTI~SENSE: yes (xi) SEQUENCE DESCRIPTION: SE~ ID NO: l9 (2) INFORMATION FOR SEQ ID NO: 20:
~i) SEQUENCE CNARACTERISTICS:
(A) LENGTH: 20 t~) TYPE: nucleic acid (C) STRANDE~NESS: single ~D).TOPOLOGY: lin~ar (iv) ANTI-SENSE: yes :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20~
TGGAATCAGA CACAAGCCGT 2 0 `-~ . .
(2) INFORMATION FOR SEQ ID NO: 2l: ~.
(i~ SEQUENCE CHARA~TERISTICS~
~` ~(A) LENGTH: 20 (B) TYPE: nucleic acid (C) ST ~ DEDNESS: single : : (D) TOPOLV&Y: linear . ~:
:: (iv~ ANTI-SENSE: yes ; :~
~ .
` :(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CATCTTGCGC:GCGGGG~GCC 20 :-.
~ -;
;..
. .
2132~9~
~2) INFO~MATION FOR SEQ ID NO: 22: ;-(i) SEQUENCE C~ARACTERISTICS: .
(A) LENGTH: 20 (B) TYPE: nucleic acid ¦ -(C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear -(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
TGC&CGCGGG GAGCCGGAGC 20 -.
(2) INFOR~ATION FOR SEQ ID NO: 23:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 ~B) TYPE: nucleic acid (C) STRANDEDNESS: single .~;
(D) TOPO~OGY: linear .:
(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTIQN: SEQ ID NO: 23: ,, (2)~ INFORMATION FOR SEQ ID NO: 24: -(i) SEQUENCE CEARACTERISTICS: .
(A) LENGTH: 20 ~B) TYPE: nucleic acid j STR~NDEDNE5S: single :~
: ~D) TOPOLOGY: linear :~ ~ (iv) ANTI-SENSE:~ yes (xi) SEQUENCE~DES~RIPTION: SEQ ID NO: 24:
, .
~ CTCTCCTCGC CCTCCGT~GG 20 s .
~ `
WO93/19203 PCT/U~g3/02213 3 ~ 09 1 ~ 36 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STR~NDEDNESS: single ~D) TOPOLOGY: linear (iv) ANTI-S~NSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
(2) INFORMATION FOR SEQ ID no: 26:
(i) SEQUENCE CH~RACTERISTICS:
, (A) LENGTH: 20 : (B) TYPE: nucleic acid : ~ (C~ STRANDEDNESS: single ~:~ (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUEN~E DESCRIPTION: SEQ ID NO: 26:
: : : AAAGGCCTCT AAGACA~GCT 20 ; (2) INFO~M~TION FOR SEQ ID~NO: 27: :
i) SEQUENCE~CHARACTERISTICS~
) LENGTH: 20 ~ ;
, (B~ TYPE:~nucleic acid .-STRANDEDNESS: single D) TOPOLOGY: linear (iv)~ANTI-SENS~: yes (xi)~ SEQUENCE DESCRIPTION: SEQ ID NO: 27: ~, ; ~ GCCAGCATGT GCACCGTGAA 20 ~ W O 93/19203 2 1 ~ 2 ~ 9 4 PCr/US93/02213 (2) INFORUS~TION FOR SE~ ID NO: 28: :~
(i) SEQU ~ CE CH~RACTEDRISTIC5: `
(A) ~ENGTH: 20 ~:
(B) TYPE: nucleic acid ~ .
~C) STRU~NDEDNESS: single (D) TOPO ~ GY: linear (iv) ~NTI-SENSE: y~s (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
ACACCCC~GG CTC~CGATG 20 ~2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CCG~GCTTA CTCAC~TTT 20 ~-~
(2) INFORUS~TION~ FOR SEQ. ID NO: 30:
(i) SEQUENCE CH~R~CTERISTICS: :
: (A) LENGT~: 20 (B) TYPE: nucleic acid C) STRANDEDNESS: single : ~ (D~ TOPOLOGY:~linear ~ ;
(i~) ANTI-SENSE: yes~
,.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ACTTAGCTCT~TGACTTCGGG 20 ~ ~ ' :~ :
W ~ PCT/US~3/D2213 - 38 - .
(2) INFO~MATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 20 (B) TYPE: nu~leic acid --(C) STRANDEDNE5S: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) 5EQUENCE DESCRIPTION: SEQ ID NO: 31:
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ~ .
(Cj STRANDEDNESS: single ~ -(D~ TOPOLOGY: linear (iv) AN~I-SENSE:~yes : (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
TTTTATTTT GAGCA~TTC 20 .
(2) INFO~MATION FOR~SEQ;ID NO: 33~
i) SEQ~ENCE~CHARACTERISTICS: : ~?
A)~LENGTH~;~20 (B) TYPE: nucleic ~cid : ~ . -.
STRANDEDNESS: single (D)~TOPOLOGY:~linear :(iv~;~ANTI-SENSE:::yes~
(xi)~SEQUENCE~DESCRIPTION: SEQ ID NO: 33: ~3 '' TTTGGGATG A~GGTGA~CA 20 ~ ;
WO93/19203 ~1~ 2 0 9 ~1 PCT/US93/02213 - 39 - :
(2) INFOP~ATIQN FOR SEQ ID NO: ~4:
(i3 SEQUENCE C ~ CTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv~ ANTI-SENSE: ye~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CCCATTCCCA CAGGCCTGAG 23 `
~2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B~ TYP~: nucleic acid .
(C) STRANDEDN~SS: single :~
(D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 1 ~ CGGAGCGCGC CAGGCAGGGA 20 :-:: ~2) INFORMA~ION FOR SEQ ID NO: 36: ~ ..
SEQUENCE~CHARACTERISTICS:
A): LENGTH: 20 (~j TYPE: nucleic acid (C) STRANDEDNESS: single , ~:
; (D) I'OPOLOGY: linear iv) ~NTI-~ENSE:~ yes (xi) :SEQUENCE DESC~IPTION: SEQ ID NO: 36:
: CCTTTTCGCA GACCAGCC~T 20 :
:~ : : :,-.
P~T~US93/~22~3 WO g3~19203 2~3 2 9 ~ - 40 (2) INFORMAT~ON FOR SEQ ID NO: 37:
(i) SEQUENCE CHAR~CTERXSTICS:
(~) LENGTH: 20 (B) ~YPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
:GGCCCCAG~A ACG~AGCAGG 20 (2) INFORMATION FOR SEQ ID NO: 3:8:
(i) 5EQUENCE C~ARACTERISTICS:
: (~3 LENGTH: 20 (.B) TYPE: nucleic acid (C): STRANDEDNESS: single (D) TOPOLOGY: linear~ ~:
~iv) ANTI-SENSE: yes ~ (xi) SEQUENCE DESCRIPTION::~;SEQ ID NO: 38:
:; : GGATCCTGCC TTTC~TTGGGG 20 ~
.:
(2) INFORM~TION FOR SEQ ID NO: 39 SEQUENCE CHARACTERISTICS:~
A)~LENGTH: 20 (B);T~PE: nucleic ac;id (Cj~ STRANDEDN~SS: single D)~TOPOLQGY:jlinear $v)::ANTI-5ENSE: yes~
(xi)~ SEQUENCE DESC:IPTION: SEQ ID NO: 3~:
CAGCCATGGC CC QCAAACG 20~
WO93/19~03 2 1 3 2 0 ~ ~ PCT/~S93/0 ~13 (2) INFORMATION FOR SE~ ID NO: 40:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv) ANTI-SENSE: yes (xi~ SEQUENCE DESCRIPTION: SE~ ID NO: 40:
(2) INFORMATIQN FOR SE~ ID NO: 41: :
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B~ TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: 1inear (iv) ANTI-SENSE: yes (xi) SE~UENCE DESCRIPTION: SEQ ID NO: 41:
~2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTER~STICS:
(A) LENGTH~: 20 tB) TYPE: n,ucleic acid ~C) STRANDEDMESS: ~ingle (D)~TOPOLOGYt linear (iv) ANTI-SENSE:;yes ,~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GCCTGCTTCG CAGCGG~AGA 20 ::
W093/~9~03 ~CT/US93/02213 I . .
~3~ 42 ~
- (2) INFOR~ATION FOR 5EQ ID NQ: 43:
(i) SEQUENCE CHARACTERISTICS~
(A~ LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) AN~I-SENSE: ye~
~xi) ~EQUENCE DESCRIPTION: SEQ ID NO: 43: -~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 : ~ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear : (i~) ANTI-SE~SE: ye~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
GTCC GTCTC~AGGCCAGCCC 20 (2) INFO~MATION FO~ SEQ ID NO: 45.
r (i) SEQUENCE CHA~ACTERISTICS: ,,.
; (A) LENGTH: 20 (B) TYPE~ nucleic acid ~.
(C) STRANDEDNE5S: single (D) TOPOLOGY: linear .
~:: (i~) ANTI-S~NSE: yes ~ .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
:C~T~C~GAT GCGGACCCTC 20 ~.
. ~
~ : , :
W093/19203 2 1 3 2 0 ~ ~ PCTIUS93/02213 `~
(2) INFO~MATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A3 LENGTH: 20 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: sîngle (D) TOPOLOGY: linear (iv) ANTI-SENSE: y~s (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
~2) INFORMATION FOR SEQ ID NO: 47:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ~N~I-SENSE: yes (xi) SEQUENCE DESCRIPTION: SE~ ID NO: 47: 2 ~: (2) INFORMA~ION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A);LENGTH: 20 (B) TYPE: nucleic acid (Cj STRANDEDNESS: single (D)~ ~OPOLOGY: linear iv) ANTI-SEN5E: yes ~(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 4~:
TTCCTTT~GG~TTCTCGTGCC 20 (2) INFORMATION FOR SEQ ID NO: 49:
(i~ SEQUENCE C~ARACTERISTIC5:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDMES~: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2~ INFORMATION FQR SEQ I~ NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: S0:
(2) INFO~M~TION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) T~PE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY:~linear ~(iv) ANTI SENSE: ye~
~: :
(xi) SEQUENCE DESCRIPTION: ~EQ ID NO: 51:
WO 93/19~03 ~ L PCI/US93/02~13 45 - :
( 2 ) INFORMATION FOR SEQ ID NO: S2:
( i) SEQUENCE CHAR~CT:~ISTICS:
( A) LENt;TH: 2 0 (B) TYPE: nucleic acid ( C ) ST~NDEI:~NESS: s ingle (D) TOPOIJOGY: linear `. ( iv) ANTI-SENSE: rlo a (Xi) SEQUENCE DESCRIPTION: SEQ ID N0: 52 ;~ TAACATACAT CATGGGGCCG 20 ;1 _(2) INFO~MATION FOR SEQ ID NO: S3 ( i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 20 '` . (B) TYPE: nucleic acid 3 ( C) STRANDEDNESS: single )) TOPOLOGY: 1inear ,~
( iv) ANTI~5ENSE: no ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : S 3:
; GGTTTT~CCA T~GGTTCTG& 20 (2) INFORM~TION FOR SEQ ID N0: 54-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ! . ( C) ST~ANDEDNESS: sing~e D~ TOPOLOGY: linear (iv) AMTI-SEMSE: no ~ (Xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 54:
J GTTACACAGG GACTC~ACCC 20 rl ';' .'11
NO: 53) and 4608 (SEQ ID NO: 54~. A549 cells were treat~d for four hours with oligonucleotide (either 400 nM or 300 nM) and ,. .
DOTMA, washed, and allowed to recover for 48 hours. Cells were then treated again with oligonucleotide (250 nM) and DOTMA, ~-15 washed and allowed to recover for a further 20 hours. Cell `-extracts were electrophoresed, transferred to membrane and .~-blocked as described in Example 3. ~he membrane was then , .
treatsd overni~ht with either of the following antibodies diluted in 0.5% nonfat milk in TBS: anti-PKC-~, B or 20 :monoclonal (Upstate Biochemicals, Inc.3, 1 ~g/ml; anti PKC-~ or ..
-~ polyclonal (Gibco BRL~ 2000 dilution; anti-PXC-~ [Huwiler et al., Bioche~. J. 199g, 279, 441~445], 1:4000 dilution; or L ~=
anti-PKC~ 4000 dilution. Anti~ody incubation was followed .~.
by three washes in TBS containing 0.1~ nonfat milk and the 25 memb~ane was then incubated with 5 ~Ci 1~sI-goat anti~rabbit or anti-mouse antibody (ICN Radiochemicals, Irvine CA) for one :
hour. Membranes were washed extensively and the labelled .--~
proteins were visualized and quantitated using a phosphorimager (MO1QCU1ar Dynamics, CAj~
'` 30 Analysis of the PKC isozymes expressed after -: ~ligonucleotide ~reatment revealed that le~els of PKC~ and ~:
-~ were un hanged by treatment with either of the antisense oligonucleotides or their scrambled controls. The antisense !~^
oligonucleotides decreased PKC-~ expression by 70-r80%, while scrambled controls had no effect.
,.
1, .
W093/~9203 PCT/US93/02213 ~ampl~ 10 Ef~ect of oligonualeoti~s3 on A549 cell proli~ratio~
A549 cells were plated in 6-well plates at a concentration of 4000 cells per well~ After 24 hours cells 5 were washed three times in.DMEM and then oligonucleotides were added to t~e required concentration in the presence of 20 ~g/ml DOTMA for four hours. Cel:ls were then washed once in DMEM plus 5~ FCS and allowed to grow in DME~ plus 5% FCS for a further 72 hours. At this time cells were washed twice in PBS, removed 0 from the dishes with trypsin and counted with a hemocytometer.
Inhibition of A549 cell proliferation by ISIS 3S21 and 3527:was seen after a single oligonucleotide dose of 250-500 nM. This is æhown in Figure 5.
~xample ll I~hibition of PRC~ by chim~ric ~nti~ns~
oligonu¢leoti~es Chimeric phosphorothioate oligonucleotides having "deoxy : gapæ:" of 4 to 8~deoxynucleotide~ in an otherwise 2'-0-methyl oli~onucleotide were tested for ability to decrease PKC~ m~NA
levels as:des~ribed in Example 8. Chimeric oligonucleotides 20 ~were identical in sequence to ISIS 3521 (SE~ ID N0:2) ISIS 3522 (SEQ ~ID N0: 3) and~ISIS 3527 lSEQ ID N0:5). Chimeric ` oligonucleotides are:shown in Table 7:
:
: :
WO 93/19203 2 1 3 ~ O ~ ~ PC~ /us93/o22l3 ~
PKcA GAPPED OLI&ONUCLEOTIDES
Boldl indicates 2 ' -O-methyl nucleotides All oligonuc:leotide are phosphorothioat2s OLIGO SEQUl :NCIS DESCRIPTION SEQ ID NO
3521 GTTCTCGCTGGTGAGTTTCA Full P=S
5357 ~ C$C:GCTGGTGAGTTTCA Full P=S 8-deoxy gap 2 S361 G~T~CTC~3CTGGTGAGT~TC~ Full P--S 6-deoxy gap 2 53~0 tS~CTC~3CTGGTGA~rTTC~ Full P=S 4-deoxy gap 2 10 3522 A~ACGTCAt:CCATGGTCCC Full P=S 3 535~ AAAACGTC~GCCA~GGq!CCC Full P=S 8-deoxy gap 3 5350 ~CG~CAGCCA~GTCCC Full P=S 6-deoxy gap 3 S351 AAa;~CG~rCA~;CCP~GG~t~CC Full P-S 4-deoxy gap 3 3527 GAG~CCCTGAACAGTTGATC Full P=S 5 15 5240 ~;~GACCCTGAAC~ :;TTG~TC Full P=S 8~deoxy gap 5 5208 GAG;~CS~C~GAAC.~TTGATC Full P=S 6-deoxy gap 5 5038 GAG~CCCTGAACAt~TTGATC Full P=S 4-deoxy gap 5 The 8-deoxy ga~pped oligonucleotides were able to lower PKC~ ~ NA le~rels by at least 85%. Activity of 8-deoxy gapped 20 oligonucleotides compared to ungapped oligonucleotides having the same sequence is shown in Figure 6A, 6B and 6C. Two of the 6-deoxy ~apped oligonucleotides ( ISXS 53 61, SEQ ID NO: 2 . and ISIS 5350, SEQ ID NO:3 ) were able to lower PKCc~ mRNA by greater than 50%, and one of the 4-~eoxy gapped 25 oligonucleotides (I5IS 3522, SEQ I1:) NO: 3) was able to inhibit PKCa~ mRNA by approximately 85%. Activity vs. d.eoxy gap length f or oligos having SEQ ID NC): 2, SEQ ID NO: 3 ~nd SEQ ID ~O: 5 is shown in Figure 7.
I , i , , I . ~ j .
~a~pl~ 12 Oligonucleot~e tr~t~t of hu~a~ tu~or eell~
~u~a ~ic~ 'f Human Iung carcinoma A549 cells were harves~ed and 5 x : 106 cells were inje~ted subcutaneously into the inside hind leg of nude mice. Palpable tumors davelop in approximately one month~ Antisense oligonucleotides ISIS 3521 and 3527 are adminis~ered to mice intraperitoneally at two doses, 1 and 10 :
::
mg/kg body weight, every other day for six wee3cs. Mice are monitored for tumor gro~rth and after six weeks the t~mors are excised and the expression of PKCc~ :~s de~ermined ~y Northern blot and immunoblot.
Although preferred embodiments of the invention have been described herein~
it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims.
-~ : :
.
: ~ ' : .
-SEQUENCE LISTING
(1) GENERAL INFORMATION:
(.i) APPLICANT: Nicholas Dean, C. Frank Bennett (ii) TITLE OF INVENTION: Oligonucleotide Modulation of Protein Kinase C
(iii) NUMBER OF SEQUENCES: 54 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Woodcock Washburn Kurtz Mackiewicz & Norris (B) STREET: One Liberty Place - 46th Floor ~ (C) CITY: Philadelphia (D) STATE: PA
(E~ COUNTRY: USA
(F) ZIP: 19103 (v) COMPUTER~READABLE FORM:
(A~ MEDI~M TYPE: DISKETTE, 3.5 INCH, 1.44 Mb STORAGE
(B) COMPUTER: IBM PS/2 (C) OPERATING SYSTEM: PC-DOS
~D) SOFTWARE: WORDPERFECT 5.0 ;~ (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: n/a (B) FILING DATE: herewith (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A3 APPLICATION NUMBER: 852,852 (B) FILING:DATE: March 16, 1992 ~: ~viii)~ATToRNEY/AGENT INFORMATION:
; ~ ~ (A):N~ME: Jane Massey Licata (B) REGISTRATION NUMBER: 32,257 ,.,r~
;`::: :: `:
: : :
W093/19~03 PCT/U~93/02~13 ~3~09~ - 28 -(C) REFERENOE/DOCKET NUMBER: ISIS-0872 (ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (215) 568~3l00 (B) TELEFAX: (2l5) 568-3439 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CXARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS~ single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
CCCCA~CCAC CTCTTGCTCC 20 (2) INFORMATION FOR SEQ ID NO: 2:
~i) SEQUENCE CHARACTE~ISTICS:
~: (A) LENGTH:~20 ~B) TYPE: nucleic acid (C) STRANDEDNESS: single : ~ (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes ~(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
(2~ INFORMATION FOR ~EQ;ID NO: 3:
~k ~ ~ I
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGTH:~:20 (B) TYPE: nucleic acid : ~ :
~ (C)~ STRANDEDNESS:~single ::: : : : ` :
~ (D) TOPOLOGY::linear ~ ~ .
WO 93/19203 PCr/US93/OZ213 ( iv) ANTIWSENSEq yes ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 3:
~ GTCAG CCATGGTCCC 2 0 ( 2 ) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACrERISTï::S:
( A ) LENGTH: 2 0 (B) TYPE: nuclei :: acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ( iv) ANTI~SENSE: yes (xi) SEQUE~CE DESCRIPTION: SEQ Il) NO: 4:
GGATTCACTT CCACTGCGGÇ: 2 0 t 2 ) INFORM~TION FOR SEQ ID NO: 5 ~ ;
( i ) SEQUENCE C~ACTERISTICS:
( ~ ) LENGTEI: 2 O
(B) TYPE: nucleic acid ~ ) STRA~DEDNESS: single ( D`) TOPOLOGY: l inear t i~) ANTI-SENSE: yes ( xi ) S~QUENCE: DESC~IPTION: SEQ ID NO: 5:
GACCCTGA P.CP~GTT :;ATC 2 û
.
( 2 ) XNFORMATIOM FQR ^SEQ TD NO: 6:
(i) 5EQUEN~E C~ARACTERISTICS:
1~ ' .
tA) LENGTH: 20 s (B) TYPE. nucleic ac:id (C) ST~DEDNESS: single ( D ~ TOPOLOGY: l inear ( iv~) ANTI~5ENSE: yes W093~19203 PCT/US93tO2213 ~ 0 9 ~ 30 _ ' (xi) SEQUEMCE DESCRIPTION: SEQ ID NO: 6:
CCCG~GAA~A CGT~AGCCAT 20 (2) INFO~MATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) ST ~ DEDNESS: single (D3 TOPOLOGY: linear (iv) ~NTI-SENSE: yes : (xi) SEQUENCE DESCRIP~ION: SEQ ID NO: 7:
(2) INFORMATION FOR SEQ ID NO: 8:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B~ TYPE: nucleic acid (C~ STR~NDEDNESS: single : ~ . (D) TOPOLOGY: linear (iv3 ~NTI-SENSE: yes : ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
(2) INFORMATION FOR:SEQ ID NO: 9:
SEQUENCE:C~ARACTERISTICS:
(A)~LENGTH~:~20 ~B)~;~TYPE: nucleic acid (C~) STRANDEDNESS~: single (D)~TOPOLOGY: linear (iv): ~NTI-SENS ^ yes - ; (xi~) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
.
, ~
W093/19203 2 ~ 3 ~ ~ 9 ~ PCT/US93/02213 1 ' - 3~ ~ :
GCAG~GGCTG GGGACATTGA 20 (2) INFORMATION FOR SEQ ID NO l0: ~ -(i) SEQUENCE CH~RACTERISTICS:
~A) LEN&TH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: l0:
t2) INFORMATION FOR SEQ ID NO: ll: ` ~.
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 ; ~B~ TYPE: nucleic acid (C3 ST~ANDEDNESS: single (D) TOPOLOGY: linear ~iv) ANTI~SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: ll:
: CACTGCGGGG AGGGCTGGGG 20 (2) INFQRMATION FOR SEQ ID NO: 12: i (i) SEQUENCE CHARACTERISTICS: :
(~) LENGTH: 20 .
tB) TYPE: nucleic acid (C) STRANDEDNESS: sin~le (D) TOPOhOGY~: linear ~iv) ANTI-SENSE- yes .
:~ ~ (xi~ SEQUENCE~DESCRIPTIVN: SEQ ID NO: 12:
WO93/l9203 PCT/US93/02213 :
~o94 - 32 (~) ORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv3 ANTI SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:
-~2) INFORMATION FOR~SEQ ID NO: 14 (i) SEQUENCE CHARACTERISTICS~
(A) LENGTH: 20 (B) TYPE: nucleic acid:
(C) 5TRANDEDNESS: single , : (D) TOPOLOGY: linear ~iv) ~NTI-SEN~E: yes (xi) SEQUEN~E DESCRIPTION: SEQ ID NO 14: . ~' A~GAGAGAGA CCCTGAACAG 20 :
(2)~ INFORMATION~FOR SEQ ID NO: lS~
SEQUENCE CHARACTERISTICS~
(A) ~ENGT~: 20~
B) TYPE: nucleic acid (C) STRANDEDNESS: single ~ q (D)~TOPOLOGY~ linear~
(iv)~ANTI-SENSE:~yes~
SEQUEN~E~E~RIPTION~ SEQ ID~:NO::15 GATAATGTTC~TT6GTTGTAA 20 W093/19203 2~32~ PCI/US93/02~13 (2) INFORMATION FOR SEQ ID NC): 16:
i ) SEQtJENC13 C~CTERISTICS
(A~ L:~SNGTH: 20 (B~ q~YPE: nucleic acid t ;~
( C) STRANDED~ESS: single ` .
~ D ) TOP~;)LO :;Y: 1 inear - . .
( iv~ ANTI SENSE: yes ~xi) SEQUENCE r)ESCRIPTION: SE~ ID NO: 16:
ATG~:;GGTGCA CA~CTGGGG 2 0 . ~;
. . , .2 ) INFORMATION FOR EQ ID NO : 17:
i ) SEQUENCE CHARACTERISTICS:
(A) ~ENt;TH: 20 (B) TYPE: nucleic acid . ~ .
(C) ST~DEDNESS: single ~-(D) ~OPOLOGY:~ linear .
~ iv) ANT~-SENSE: yes . . ; ;~
(xi) SEQUENCE DESCRIPTIC1N: S~Q ïD NO: 17:
~TCAGCCATG GTCCCCCCCC 2 0 : .
( 2 ) INFORM~TION FOR SEQ ID NO: 18:
( i) SEQIJ:E:NCE CHARACTERISTICS:
( A ~ LENGTH : 2 0 ~;
~.
(B) TYP~: nuclei :: acid i ( C) STRANDEDNESS: siYIgle (D) TOPOLOGY: linear ~, ( iv~ ANTI~SENSE: y~s : ~
: 1 : ~ ( Xi ) SEQUENC:E DESCRIPTI~ON: SEQ ID NC): 18: ~
:
~ 3 ~ ~ 3~ ! :
(2) INFORMAT~ON FOR SEQ ID NO: l9:
~i~ SEQUENCE C~ARACTERISTICS~
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv) ~NTI~SENSE: yes (xi) SEQUENCE DESCRIPTION: SE~ ID NO: l9 (2) INFORMATION FOR SEQ ID NO: 20:
~i) SEQUENCE CNARACTERISTICS:
(A) LENGTH: 20 t~) TYPE: nucleic acid (C) STRANDE~NESS: single ~D).TOPOLOGY: lin~ar (iv) ANTI-SENSE: yes :
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20~
TGGAATCAGA CACAAGCCGT 2 0 `-~ . .
(2) INFORMATION FOR SEQ ID NO: 2l: ~.
(i~ SEQUENCE CHARA~TERISTICS~
~` ~(A) LENGTH: 20 (B) TYPE: nucleic acid (C) ST ~ DEDNESS: single : : (D) TOPOLV&Y: linear . ~:
:: (iv~ ANTI-SENSE: yes ; :~
~ .
` :(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 21:
CATCTTGCGC:GCGGGG~GCC 20 :-.
~ -;
;..
. .
2132~9~
~2) INFO~MATION FOR SEQ ID NO: 22: ;-(i) SEQUENCE C~ARACTERISTICS: .
(A) LENGTH: 20 (B) TYPE: nucleic acid ¦ -(C) STRANDEDNESS: ~ingle (D) TOPOLOGY: linear -(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22:
TGC&CGCGGG GAGCCGGAGC 20 -.
(2) INFOR~ATION FOR SEQ ID NO: 23:
(i) SEQUENCE C~ARACTERISTICS:
(A) LENGTH: 20 ~B) TYPE: nucleic acid (C) STRANDEDNESS: single .~;
(D) TOPO~OGY: linear .:
(iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTIQN: SEQ ID NO: 23: ,, (2)~ INFORMATION FOR SEQ ID NO: 24: -(i) SEQUENCE CEARACTERISTICS: .
(A) LENGTH: 20 ~B) TYPE: nucleic acid j STR~NDEDNE5S: single :~
: ~D) TOPOLOGY: linear :~ ~ (iv) ANTI-SENSE:~ yes (xi) SEQUENCE~DES~RIPTION: SEQ ID NO: 24:
, .
~ CTCTCCTCGC CCTCCGT~GG 20 s .
~ `
WO93/19203 PCT/U~g3/02213 3 ~ 09 1 ~ 36 (2) INFORMATION FOR SEQ ID NO: 25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STR~NDEDNESS: single ~D) TOPOLOGY: linear (iv) ANTI-S~NSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:
(2) INFORMATION FOR SEQ ID no: 26:
(i) SEQUENCE CH~RACTERISTICS:
, (A) LENGTH: 20 : (B) TYPE: nucleic acid : ~ (C~ STRANDEDNESS: single ~:~ (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUEN~E DESCRIPTION: SEQ ID NO: 26:
: : : AAAGGCCTCT AAGACA~GCT 20 ; (2) INFO~M~TION FOR SEQ ID~NO: 27: :
i) SEQUENCE~CHARACTERISTICS~
) LENGTH: 20 ~ ;
, (B~ TYPE:~nucleic acid .-STRANDEDNESS: single D) TOPOLOGY: linear (iv)~ANTI-SENS~: yes (xi)~ SEQUENCE DESCRIPTION: SEQ ID NO: 27: ~, ; ~ GCCAGCATGT GCACCGTGAA 20 ~ W O 93/19203 2 1 ~ 2 ~ 9 4 PCr/US93/02213 (2) INFORUS~TION FOR SE~ ID NO: 28: :~
(i) SEQU ~ CE CH~RACTEDRISTIC5: `
(A) ~ENGTH: 20 ~:
(B) TYPE: nucleic acid ~ .
~C) STRU~NDEDNESS: single (D) TOPO ~ GY: linear (iv) ~NTI-SENSE: y~s (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:
ACACCCC~GG CTC~CGATG 20 ~2) INFORMATION FOR SEQ ID NO: 29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:
CCG~GCTTA CTCAC~TTT 20 ~-~
(2) INFORUS~TION~ FOR SEQ. ID NO: 30:
(i) SEQUENCE CH~R~CTERISTICS: :
: (A) LENGT~: 20 (B) TYPE: nucleic acid C) STRANDEDNESS: single : ~ (D~ TOPOLOGY:~linear ~ ;
(i~) ANTI-SENSE: yes~
,.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30:
ACTTAGCTCT~TGACTTCGGG 20 ~ ~ ' :~ :
W ~ PCT/US~3/D2213 - 38 - .
(2) INFO~MATION FOR SEQ ID NO: 31:
(i) SEQUENCE CHAR~CTERISTICS:
(A) LENGTH: 20 (B) TYPE: nu~leic acid --(C) STRANDEDNE5S: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) 5EQUENCE DESCRIPTION: SEQ ID NO: 31:
(2) INFORMATION FOR SEQ ID NO: 32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ~ .
(Cj STRANDEDNESS: single ~ -(D~ TOPOLOGY: linear (iv) AN~I-SENSE:~yes : (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:
TTTTATTTT GAGCA~TTC 20 .
(2) INFO~MATION FOR~SEQ;ID NO: 33~
i) SEQ~ENCE~CHARACTERISTICS: : ~?
A)~LENGTH~;~20 (B) TYPE: nucleic ~cid : ~ . -.
STRANDEDNESS: single (D)~TOPOLOGY:~linear :(iv~;~ANTI-SENSE:::yes~
(xi)~SEQUENCE~DESCRIPTION: SEQ ID NO: 33: ~3 '' TTTGGGATG A~GGTGA~CA 20 ~ ;
WO93/19203 ~1~ 2 0 9 ~1 PCT/US93/02213 - 39 - :
(2) INFOP~ATIQN FOR SEQ ID NO: ~4:
(i3 SEQUENCE C ~ CTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv~ ANTI-SENSE: ye~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
CCCATTCCCA CAGGCCTGAG 23 `
~2) INFORMATION FOR SEQ ID NO: 35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B~ TYP~: nucleic acid .
(C) STRANDEDN~SS: single :~
(D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: 1 ~ CGGAGCGCGC CAGGCAGGGA 20 :-:: ~2) INFORMA~ION FOR SEQ ID NO: 36: ~ ..
SEQUENCE~CHARACTERISTICS:
A): LENGTH: 20 (~j TYPE: nucleic acid (C) STRANDEDNESS: single , ~:
; (D) I'OPOLOGY: linear iv) ~NTI-~ENSE:~ yes (xi) :SEQUENCE DESC~IPTION: SEQ ID NO: 36:
: CCTTTTCGCA GACCAGCC~T 20 :
:~ : : :,-.
P~T~US93/~22~3 WO g3~19203 2~3 2 9 ~ - 40 (2) INFORMAT~ON FOR SEQ ID NO: 37:
(i) SEQUENCE CHAR~CTERXSTICS:
(~) LENGTH: 20 (B) ~YPE: nucleic acid (C) STRANDEDNESS: single ~D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:
:GGCCCCAG~A ACG~AGCAGG 20 (2) INFORMATION FOR SEQ ID NO: 3:8:
(i) 5EQUENCE C~ARACTERISTICS:
: (~3 LENGTH: 20 (.B) TYPE: nucleic acid (C): STRANDEDNESS: single (D) TOPOLOGY: linear~ ~:
~iv) ANTI-SENSE: yes ~ (xi) SEQUENCE DESCRIPTION::~;SEQ ID NO: 38:
:; : GGATCCTGCC TTTC~TTGGGG 20 ~
.:
(2) INFORM~TION FOR SEQ ID NO: 39 SEQUENCE CHARACTERISTICS:~
A)~LENGTH: 20 (B);T~PE: nucleic ac;id (Cj~ STRANDEDN~SS: single D)~TOPOLQGY:jlinear $v)::ANTI-5ENSE: yes~
(xi)~ SEQUENCE DESC:IPTION: SEQ ID NO: 3~:
CAGCCATGGC CC QCAAACG 20~
WO93/19~03 2 1 3 2 0 ~ ~ PCT/~S93/0 ~13 (2) INFORMATION FOR SE~ ID NO: 40:
(i) SEQUENCE CH~RACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear ~iv) ANTI-SENSE: yes (xi~ SEQUENCE DESCRIPTION: SE~ ID NO: 40:
(2) INFORMATIQN FOR SE~ ID NO: 41: :
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B~ TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: 1inear (iv) ANTI-SENSE: yes (xi) SE~UENCE DESCRIPTION: SEQ ID NO: 41:
~2) INFORMATION FOR SEQ ID NO: 42:
(i) SEQUENCE CHARACTER~STICS:
(A) LENGTH~: 20 tB) TYPE: n,ucleic acid ~C) STRANDEDMESS: ~ingle (D)~TOPOLOGYt linear (iv) ANTI-SENSE:;yes ,~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:
GCCTGCTTCG CAGCGG~AGA 20 ::
W093/~9~03 ~CT/US93/02213 I . .
~3~ 42 ~
- (2) INFOR~ATION FOR 5EQ ID NQ: 43:
(i) SEQUENCE CHARACTERISTICS~
(A~ LENGTH: 20 (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) AN~I-SENSE: ye~
~xi) ~EQUENCE DESCRIPTION: SEQ ID NO: 43: -~2) INFORMATION FOR SEQ ID NO: 44:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 20 : ~ (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear : (i~) ANTI-SE~SE: ye~
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44:
GTCC GTCTC~AGGCCAGCCC 20 (2) INFO~MATION FO~ SEQ ID NO: 45.
r (i) SEQUENCE CHA~ACTERISTICS: ,,.
; (A) LENGTH: 20 (B) TYPE~ nucleic acid ~.
(C) STRANDEDNE5S: single (D) TOPOLOGY: linear .
~:: (i~) ANTI-S~NSE: yes ~ .
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:
:C~T~C~GAT GCGGACCCTC 20 ~.
. ~
~ : , :
W093/19203 2 1 3 2 0 ~ ~ PCTIUS93/02213 `~
(2) INFO~MATION FOR SEQ ID NO: 46:
(i) SEQUENCE CHARACTERISTICS:
(A3 LENGTH: 20 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: sîngle (D) TOPOLOGY: linear (iv) ANTI-SENSE: y~s (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:
~2) INFORMATION FOR SEQ ID NO: 47:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ~N~I-SENSE: yes (xi) SEQUENCE DESCRIPTION: SE~ ID NO: 47: 2 ~: (2) INFORMA~ION FOR SEQ ID NO: 48:
(i) SEQUENCE CHARACTERISTICS:
(A);LENGTH: 20 (B) TYPE: nucleic acid (Cj STRANDEDNESS: single (D)~ ~OPOLOGY: linear iv) ANTI-SEN5E: yes ~(xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 4~:
TTCCTTT~GG~TTCTCGTGCC 20 (2) INFORMATION FOR SEQ ID NO: 49:
(i~ SEQUENCE C~ARACTERISTIC5:
(A) LENGTH: 20 (B) TYPE: nucleic acid (C) STRANDEDMES~: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:
(2~ INFORMATION FQR SEQ I~ NO: 50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 ~B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (iv) ANTI-SENSE: yes~
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: S0:
(2) INFO~M~TION FOR SEQ ID NO: 51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) T~PE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY:~linear ~(iv) ANTI SENSE: ye~
~: :
(xi) SEQUENCE DESCRIPTION: ~EQ ID NO: 51:
WO 93/19~03 ~ L PCI/US93/02~13 45 - :
( 2 ) INFORMATION FOR SEQ ID NO: S2:
( i) SEQUENCE CHAR~CT:~ISTICS:
( A) LENt;TH: 2 0 (B) TYPE: nucleic acid ( C ) ST~NDEI:~NESS: s ingle (D) TOPOIJOGY: linear `. ( iv) ANTI-SENSE: rlo a (Xi) SEQUENCE DESCRIPTION: SEQ ID N0: 52 ;~ TAACATACAT CATGGGGCCG 20 ;1 _(2) INFO~MATION FOR SEQ ID NO: S3 ( i) SEQUENCE CHARACTERISTICS:
(A) hENGTH: 20 '` . (B) TYPE: nucleic acid 3 ( C) STRANDEDNESS: single )) TOPOLOGY: 1inear ,~
( iv) ANTI~5ENSE: no ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : S 3:
; GGTTTT~CCA T~GGTTCTG& 20 (2) INFORM~TION FOR SEQ ID N0: 54-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 (B) TYPE: nucleic acid ! . ( C) ST~ANDEDNESS: sing~e D~ TOPOLOGY: linear (iv) AMTI-SEMSE: no ~ (Xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 54:
J GTTACACAGG GACTC~ACCC 20 rl ';' .'11
Claims (39)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. An oligonucleotide comprising from 5 to 50 nucleotide units specifically hybridizable with selected DNA
or RNA deriving from the PKC gene.
or RNA deriving from the PKC gene.
2. The oligonucleotide of claim 1 specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the PKC gene.
3. A pharmaceutical composition comprising an oligonucleotide of claim 1 and a pharmaceutically acceptable carrier.
4. The oligonucleotide of claim 1 wherein said DNA
or RNA encodes a particular PKC isozyme or set of isozymes.
or RNA encodes a particular PKC isozyme or set of isozymes.
5. The oligonucleotide of claim 4 wherein said DNA
or RNA encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC?
or RNA encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC?
6. The oligonucleotide of claim 5 wherein said oligonucleotide comprises one of the sequences identified in Tables 1, 2, 3, 4, 5 and 6.
7. A method of modulating the expression of PKC
comprising contacting tissues or cells containing the gene with an oligonucleotide comprising from 5 to 50 nucleotide units specifically hybridizable with selected DNA or RNA
deriving from the PKC gene.
comprising contacting tissues or cells containing the gene with an oligonucleotide comprising from 5 to 50 nucleotide units specifically hybridizable with selected DNA or RNA
deriving from the PKC gene.
8. The method of claim 7 wherein said oligonucleotide is specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the PKC gene.
9. The method of claim 7 wherein said DNA or RNA
encodes a particular PKC isozyme or set of isozymes.
encodes a particular PKC isozyme or set of isozymes.
10. The method of claim 9 wherein said DNA or RNA
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
11. The method of claim 10 wherein said oligonucleotide comprises one of the sequences identified in Tables 1, 2, 3, 4, 5 and 6.
12. The method of claim 7 wherein said oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID
NO: 4.
NO: 4.
13. A method of detecting the presence of DNA or RNA
which encodes PKC in cells or tissues comprising contacting the cells or tissues with an oligonucleotide comprising from 5 to 50 nucleotide units specifically hybridizable with said DNA or RNA, and detecting if hybridization has occurred.
which encodes PKC in cells or tissues comprising contacting the cells or tissues with an oligonucleotide comprising from 5 to 50 nucleotide units specifically hybridizable with said DNA or RNA, and detecting if hybridization has occurred.
14. The method of claim 13 wherein said oligonucleotide is specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the PKC gene.
15. The method of claim 13 wherein said DNA or RNA
encodes a particular PKC isozyme or set of isozymes.
encodes a particular PKC isozyme or set of isozymes.
16. The method of claim 15 wherein said DNA or RNA
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
17. The method of claim 16 wherein said oligonucleotide comprises one of the sequences identified in Tables 1, 2, 3, 4, 5 and 6.
18. The method of claim 13 wherein said oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3 or SEQ ID NO: 4.
NO: 3 or SEQ ID NO: 4.
19. A method of treating conditions associated with PKC comprising contacting an animal with an effective amount of an oligonucleotide comprising from 5 to 25 nucleotide units specifically hybridizable with selected DNA or RNA deriving from the PKC gene.
20. The method of claim 19 wherein said oligonucleotide is specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the PKC gene.
21. The method of claim 19 wherein said oligonucleotide is administered in a pharmaceutical composition comprising the oligonucleotide and a pharmaceutically acceptable carrier.
22. The method of claim 19 wherein said DNA or RNA
encodes a particular PKC isozyme or set of isozymes.
encodes a particular PKC isozyme or set of isozymes.
23. The method of claim 22 wherein said DNA or RNA
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
24. The method of claim 23 wherein said oligonucleotide comprises one of the sequences identified in Tables 1, 2, 3, 4, 5 and 6.
25. The method of claim 19 wherein said oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3 or SEQ ID NO: 4.
NO: 3 or SEQ ID NO: 4.
26. A method of diagnosing conditions associated with PKC comprising contacting cells or tissues or bodily fluids from an animal suspected of having a condition associated with PKC with an oligonucleotide comprising from 8 to 50 nucleotide units specifically hybridizable with selected DNA or RNA
deriving from the PKC gene, and determining if hybridization occurs.
deriving from the PKC gene, and determining if hybridization occurs.
27. The method of claim 26 wherein said oligonucleotide is specifically hybridizable with a translation initiation site, 5' untranslated region or 3' untranslated region of the PKC gene.
28. The method of claim 26 wherein said DNA or RNA
encodes a particular PKC isozyme or set of isozymes.
encodes a particular PKC isozyme or set of isozymes.
29. The method of claim 28 wherein said DNA or RNA
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
encodes one or more of the following: PKC-.alpha., PKC-.beta., PKC-.gamma., PKC-?.
30. The method of claim 29 wherein said oligonucleotide comprises one of the sequences identified in Tables 1, 2, 3, 4, 5 and 6.
31. The method of claim 26 wherein said oligonucleotide comprises SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID
NO: 3 or SEQ ID NO: 4.
NO: 3 or SEQ ID NO: 4.
32. An oligonucleotide comprising SEQ ID NO: 1.
33. A pharmaceutical composition comprising an oligonucleotide of claim 32 and a pharmaceutically acceptable carrier.
34. An oligonucleotide comprising SEQ ID NO: 2.
35. A pharmaceutical composition comprising an oligonucleotide of claim 34 and a pharmaceutically acceptable carrier.
36. An oligonucleotide comprising SEQ ID NO: 3.
37. A pharmaceutical composition comprising an oligonucleotide of claim 36 and a pharmaceutically acceptable carrier.
38. An oligonucleotide comprising SEQ ID NO: 5.
39. A pharmaceutical composition comprising an oligonucleotide of claim 38 and a pharmaceutically acceptable carrier.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US85285292A | 1992-03-16 | 1992-03-16 | |
US07/852,852 | 1992-03-16 |
Publications (1)
Publication Number | Publication Date |
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CA2132094A1 true CA2132094A1 (en) | 1993-09-30 |
Family
ID=25314390
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002132094A Abandoned CA2132094A1 (en) | 1992-03-16 | 1993-02-25 | Oligonucleotide modulation of protein kinase c |
Country Status (14)
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US (1) | US5703054A (en) |
EP (3) | EP0672180B1 (en) |
JP (1) | JP2635216B2 (en) |
KR (1) | KR0157487B1 (en) |
AT (1) | ATE212063T1 (en) |
AU (1) | AU675638B2 (en) |
CA (1) | CA2132094A1 (en) |
DE (1) | DE69331467T2 (en) |
DK (1) | DK0672180T3 (en) |
ES (1) | ES2170065T3 (en) |
FI (2) | FI112379B (en) |
HU (1) | HU217179B (en) |
NO (1) | NO943438L (en) |
WO (1) | WO1993019203A1 (en) |
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US5789573A (en) * | 1990-08-14 | 1998-08-04 | Isis Pharmaceuticals, Inc. | Antisense inhibition of ICAM-1, E-selectin, and CMV IE1/IE2 |
US7015315B1 (en) * | 1991-12-24 | 2006-03-21 | Isis Pharmaceuticals, Inc. | Gapped oligonucleotides |
US6537973B1 (en) * | 1992-03-16 | 2003-03-25 | Isis Pharmaceuticals, Inc. | Oligonucleotide inhibition of protein kinase C |
US5681747A (en) * | 1992-03-16 | 1997-10-28 | Isis Pharmaceuticals, Inc. | Nucleic acid sequences encoding protein kinase C and antisense inhibition of expression thereof |
US5922686A (en) * | 1992-03-16 | 1999-07-13 | Isis Pharmaceuticals, Inc. | Oligonucleotide modulation of protein kinase C |
US6153599A (en) * | 1992-03-16 | 2000-11-28 | Isis Pharmaceuticals, Inc. | Methoxyethoxy oligonucleotides for modulation of protein kinase C expression |
US6117847A (en) * | 1992-03-16 | 2000-09-12 | Isis Pharmaceuticals, Inc. | Oligonucleotides for enhanced modulation of protein kinase C expression |
AU713740B2 (en) * | 1992-03-16 | 1999-12-09 | Isis Pharmaceuticals, Inc. | Oligonucleotide modulation of protein kinase C |
NO180167C (en) * | 1994-09-08 | 1997-02-26 | Photocure As | Photochemical method for introducing molecules into the cytosol of cells |
US6645943B1 (en) | 1994-10-25 | 2003-11-11 | Hybridon, Inc. | Method of down-regulating gene expression |
US5744460A (en) * | 1996-03-07 | 1998-04-28 | Novartis Corporation | Combination for treatment of proliferative diseases |
AU4205597A (en) * | 1996-08-20 | 1998-03-06 | Novartis Ag | Methods for prevention of cellular proliferation and restenosis |
GB9618376D0 (en) * | 1996-09-04 | 1996-10-16 | Ciba Geigy Ag | Pharmaceutical compositions |
US6958239B2 (en) | 1996-11-21 | 2005-10-25 | Oligos Etc Inc. | Three component chimeric antisense oligonucleotides |
US5849902A (en) * | 1996-09-26 | 1998-12-15 | Oligos Etc. Inc. | Three component chimeric antisense oligonucleotides |
EP1086116A4 (en) * | 1998-05-21 | 2004-03-17 | Isis Pharmaceuticals Inc | Compositions and methods for the pulmonary delivery of nucleic acids |
EP1080225A4 (en) * | 1998-05-21 | 2004-02-04 | Isis Pharmaceuticals Inc | Compositions and methods for the pulmonary delivery of nucleic acids |
US6077709A (en) | 1998-09-29 | 2000-06-20 | Isis Pharmaceuticals Inc. | Antisense modulation of Survivin expression |
US6492111B1 (en) | 1998-11-25 | 2002-12-10 | Isis Pharmaceuticals, Inc. | In situ binary synthesis of biologically effective molecules |
DE19956568A1 (en) * | 1999-01-30 | 2000-08-17 | Roland Kreutzer | Method and medicament for inhibiting the expression of a given gene |
EP1198592A4 (en) * | 1999-07-06 | 2002-09-18 | Isis Pharmaceuticals Inc | Oligonucleotides incorporating both 2-aminoadenine and 5-substituted pyrimidines |
US6190869B1 (en) * | 1999-10-26 | 2001-02-20 | Isis Pharmaceuticals, Inc. | Antisense inhibition of protein kinase C-theta expression |
US8008071B2 (en) * | 1999-11-08 | 2011-08-30 | University Of South Florida | Compositions and methods for detecting intracellular glucose and analogs thereof |
US6852529B1 (en) * | 1999-11-08 | 2005-02-08 | University Of South Florida | Gloucose-regulated mRNA instability element |
CN1274072C (en) * | 2000-08-03 | 2006-09-06 | 松下电器产业株式会社 | Brushless motor and method of Manufacturing the brushless motor |
US20050182006A1 (en) * | 2001-05-18 | 2005-08-18 | Sirna Therapeutics, Inc | RNA interference mediated inhibition of protein kinase C alpha (PKC-alpha) gene expression using short interfering nucleic acid (siNA) |
US20030054381A1 (en) * | 2001-05-25 | 2003-03-20 | Pfizer Inc. | Genetic polymorphisms in the human neurokinin 1 receptor gene and their uses in diagnosis and treatment of diseases |
US20040014049A1 (en) * | 2002-07-19 | 2004-01-22 | Isis Pharmaceuticals Inc. | Antisense modulation of protein kinase C-iota expression |
FR2864539B1 (en) * | 2003-12-30 | 2012-10-26 | Lvmh Rech | OLIGONUCLEOTIDE AND USE THEREOF FOR MODULATING THE EXPRESSION OF BETA-1 PROTEIN-KINASE C ISOFORM AS CUTANEOUS DEPIGMENTING AGENT |
WO2024028476A1 (en) * | 2022-08-05 | 2024-02-08 | Institut National de la Santé et de la Recherche Médicale | Methods for the treatment of th2-mediated diseases |
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US4511713A (en) * | 1980-11-12 | 1985-04-16 | The Johns Hopkins University | Process for selectively controlling unwanted expression or function of foreign nucleic acids in animal or mammalian cells |
US5034506A (en) * | 1985-03-15 | 1991-07-23 | Anti-Gene Development Group | Uncharged morpholino-based polymers having achiral intersubunit linkages |
AU7851387A (en) * | 1986-08-13 | 1988-03-08 | Genetics Institute Inc. | Protein kinase c enzymes |
US5098890A (en) * | 1988-11-07 | 1992-03-24 | Temple University-Of The Commonwealth System Of Higher Education | Antisence oligonucleotides to c-myb proto-oncogene and uses thereof |
US5138045A (en) * | 1990-07-27 | 1992-08-11 | Isis Pharmaceuticals | Polyamine conjugated oligonucleotides |
EP0678029A1 (en) * | 1993-01-13 | 1995-10-25 | BURCOGLU, Arsinur | Method for using polynucleotides, oligonucleotides and derivatives thereof to treat various disease states |
US5595902A (en) * | 1993-02-06 | 1997-01-21 | Garvan Institute Of Medical Research | DNA encoding human protein kinase C (iota) |
-
1993
- 1993-02-25 EP EP93907417A patent/EP0672180B1/en not_active Expired - Lifetime
- 1993-02-25 ES ES93907417T patent/ES2170065T3/en not_active Expired - Lifetime
- 1993-02-25 AT AT93907417T patent/ATE212063T1/en not_active IP Right Cessation
- 1993-02-25 DE DE69331467T patent/DE69331467T2/en not_active Expired - Fee Related
- 1993-02-25 AU AU38025/93A patent/AU675638B2/en not_active Ceased
- 1993-02-25 CA CA002132094A patent/CA2132094A1/en not_active Abandoned
- 1993-02-25 JP JP5516616A patent/JP2635216B2/en not_active Expired - Fee Related
- 1993-02-25 WO PCT/US1993/002213 patent/WO1993019203A1/en active IP Right Grant
- 1993-02-25 KR KR1019940703206A patent/KR0157487B1/en not_active IP Right Cessation
- 1993-02-25 DK DK93907417T patent/DK0672180T3/en active
- 1993-02-25 EP EP01201830A patent/EP1126025A3/en not_active Withdrawn
- 1993-02-25 HU HU9402657A patent/HU217179B/en not_active IP Right Cessation
- 1993-02-25 EP EP02029096A patent/EP1310555A3/en not_active Withdrawn
- 1993-07-09 US US08/089,996 patent/US5703054A/en not_active Expired - Fee Related
-
1994
- 1994-09-15 FI FI944276A patent/FI112379B/en not_active IP Right Cessation
- 1994-09-15 NO NO943438A patent/NO943438L/en unknown
-
2003
- 2003-02-18 FI FI20030248A patent/FI113059B/en not_active IP Right Cessation
Also Published As
Publication number | Publication date |
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DK0672180T3 (en) | 2002-03-04 |
EP0672180B1 (en) | 2002-01-16 |
HU9402657D0 (en) | 1994-12-28 |
DE69331467T2 (en) | 2002-11-07 |
FI113059B (en) | 2004-02-27 |
JP2635216B2 (en) | 1997-07-30 |
EP0672180A4 (en) | 1997-05-02 |
FI20030248A (en) | 2003-02-18 |
FI944276A (en) | 1994-09-15 |
DE69331467D1 (en) | 2002-02-21 |
US5703054A (en) | 1997-12-30 |
ATE212063T1 (en) | 2002-02-15 |
FI944276A0 (en) | 1994-09-15 |
WO1993019203A1 (en) | 1993-09-30 |
JPH07503856A (en) | 1995-04-27 |
KR0157487B1 (en) | 1998-10-15 |
NO943438L (en) | 1994-11-11 |
FI112379B (en) | 2003-11-28 |
AU675638B2 (en) | 1997-02-13 |
EP1126025A2 (en) | 2001-08-22 |
KR950700999A (en) | 1995-02-20 |
EP1126025A3 (en) | 2002-02-13 |
AU3802593A (en) | 1993-10-21 |
HUT69939A (en) | 1995-09-28 |
HU217179B (en) | 1999-12-28 |
EP1310555A2 (en) | 2003-05-14 |
EP0672180A1 (en) | 1995-09-20 |
EP1310555A3 (en) | 2003-08-27 |
ES2170065T3 (en) | 2002-08-01 |
NO943438D0 (en) | 1994-09-15 |
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